Protocol for in vivo analysis of pre- and post-synaptic protein function in mice

Summary Studying synapses in vivo presents challenges due to the complexity of accurately targeting and visualizing specific synaptic proteins within the brain. Here, we present a protocol for in vivo analysis of pre- and post-synaptic protein function in mice. We describe steps for combining adeno-associated virus (AAV)-mediated gene transfer to manipulate specific neuron subtypes. We also describe immunofluorescence on artificial cerebrospinal fluid (ACSF)-perfused brain sections to enhance the visualization of synaptic proteins. For complete details on the use and execution of this protocol, please refer to Cramer et al.1

This protocol describes a method to investigate the function of synaptic proteins in vivo using a combination of adeno-associated virus (AAV)-mediated gene transfer and immunofluorescence.To achieve long-lasting expression of transgenes, we describe a simple technique involving neonatal intraventricular injection of genetically engineered AAVs.Compared to the common route of AAV delivery through stereotaxic surgeries, AAV injections into the neonatal ventricles are more time efficient and achieve widespread transgene expression 2,3 at the expense of spatial specificity.The resultant expression of transgenes can further be specified by the use of distinct promoters, providing an easy way to manipulate specific neuronal subtypes and their synapses throughout the brain.Particularly valuable for the investigation of synapses is the segregated manipulation of pre-synaptic or post-synaptic neurons, as it enables the investigation of pre-or post-synaptically localized proteins, as well as the site-specific function of secreted proteins localized in the synaptic cleft.To further study their function, immunofluorescence is commonly used, which enables the visualization of highly specific protein targets present at synapses using a combination of primary and secondary antibodies.Despite being a powerful technique, its effectiveness is often limited by tissue preparation methods employing transcardiac perfusion with 4% PFA, as the strength of this fixation method severely limits the immunoreactivity and detection of proteins in numerous subcellular compartments, particularly at synaptic sites.As reliable detection of synaptic proteins is crucial to understanding their function, this protocol also describes the ACSF perfusion method, which reduces the strength of tissue fixation and offers superior detection of pre-and postsynaptic proteins.This protocol is based on an initial publication investigating the function of Adamtsl3 at pre-and post-synaptic inhibitory sites using Adamtsl3 flox/flox mice described in Cramer et al., 2023. 1 The presented protocol, however, can easily be applied in various settings.Before you begin, you will need to complete the following preparatory steps.
were reviewed by the European Community Council Directives of November 24, 1986 (86/609/EEC) and approved by the cantonal veterinary office of Zurich.

Preparation one
Adeno-associated virus (AAV) selection Recombinant AAV vectors are great tools for gene transfer and have become readily available.To achieve long-term expression of a protein of interest, the following considerations should be made when selecting an AAV.
1.The relative transduction efficiency of AAV serotypes should be considered when widespread expression is desired. 2,3We have observed efficient and widespread transduction in neonatal mouse brains from the hybrid serotypes 2/6, 2/8, and 2/9, but other serotypes may be suitable, and their transduction efficiencies should be evaluated before beginning your experiment.AAVs are limited by a relatively small genome size, which allows for an insert of approximately 4.7 kb.This limits their use for large or multiple payloads.Multiple payloads will often have to be delivered using multiple AAVs, which increases experimental complexity and reduces efficiency due to incomplete co-transduction.Oversized genomes containing inserts larger than 4.7 kb can be packaged, but the efficiency of the resulting AAV will be significantly reduced.2. Nervous cell tropism varies among AAV serotypes and should be considered when cell type-specific transduction is desired.The 2/6, 2/8, and 2/9 serotypes provide excellent transduction in vivo.In addition, cell type-specific promoters should be utilized if astrocyte-, oligodendrocyte-, or neuron-specific transduction is desired, as well as the use of promoters for neuron subtype-specific transduction (excitatory, inhibitory, or exclusively parvalbumin-positive neurons for example).3. Depending on the experimental aim, constitutive or inducible transgene expression should be considered.Constitutive transgene expression is essential when investigating early periods of development, while inducible transgene expression is often desired when protein function in the adult brain is investigated and developmental defects resulting from constitute transgene expression must be avoided.Among the inducible systems, the tamoxifen (TAM)-inducible system is the most widely used, enabling temporal control over transgene expression.Also depending on the brain region/synapse of interest, a combination of AAVs should be considered when pre-synaptic or post-synaptic neuron transduction is desired.For those experiments, both neuron subtypes should be transduced simultaneously in a separate cohort to confirm the effects of neuron subtype-specific transduction.
In this protocol, we transduced neonatal Adamtsl3 flxo/flox mice with AAV2/6-CaMKIIa-Cre-ERT2 or AAV2/8-hDlx-Cre-ERT2 to ablate Adamtsl3 in adult mice either within pyramidal neurons or inhibitory interneurons respectively.On account of the well-described subcellular distribution of inhibitory synapses in the hippocampus, this combination enabled the selective investigation of presynaptic (hDlx-ERT2-Cre) and postsynaptic (CaMKIIa-Cre-ERT2) Adamtsl3 function at GABAergic synapses at CA1 pyramidal cell somata.We also injected a separate cohort with AAV2/8-hSyn1-Cre-ERT2 (not discussed) to achieve widespread transduction of all neurons to confirm the effects of neuron subtype-specific Adamtsl3 ablation.

Generation of neonatal pups for AAV injection
Intraventricular injections of genetically engineered AAV should be performed at post-natal day 0 (P0).To maximize the number of pups born for injection, it is beneficial to pair two female mice with one male mouse and set up 2 or more bleedings in parallel.Additionally, it is recommended to check the female mouse for the presence of a vaginal plug one day after mating.This plug serves as an indicator of successful mating and allows for estimation of the birth date as females will typically deliver 19 G 1 day after the appearance of the plug.Three days prior to delivery, it is also  ERT2).e. Plan to inject 2 mL of AAV for 1 pup (1 mL per ventricle).f.Keep aliquots on ice until ready for injection.
Note: Avoid additional freeze-thaw cycles of AAVs to conserve transduction efficacy.
Caution: A viral titer between 5.0 3 1012 vg/mL and 1.0 3 1013 vg/mL with an injection volume of 1 mL per ventricle is typically sufficient.Adjust if necessary.g.Prepare 5 3 500 mL aliquots of sterile distilled water to wash the syringe before the start of the procedure.Additionally, for each AAV to be injected, prepare 5 3 500 mL aliquots of sterile distilled water to wash the syringe after injection.h.Prepare a 10 mL Hamilton syringe with a 32 G needle for injection by rinsing it 5 times in each of the 5 additional Millipore water aliquots.i. Prepare an ice bucket and place an inverted petri dish on the ice to cool.j.Place dry task wipes on top of it (this will be your injection stage).
Note: Task wipes serve to protect the pup's skin from the cold dish and serve as a flat cold surface for anesthetizing the pups.
2. Transfer one pup from the warmed cage onto the cold petri dish to induce hypothermia anesthesia.a. Monitor for lack of movement and after 2-3 min, confirm anesthesia by very gently squeezing a paw. 3. Gently wipe the head of the anesthetized pup with a cotton swab soaked in 70% ethanol.
Note: Randomly select a pup from its littermates.Caution: Do not wait too long, as the pup's body temperature drops rapidly to $10 C. a. Load 2 mL of AAV2/6-CaMKIIa-Cre-ERT2 e into the Hamilton injection syringe.
Note: Assign AAVs at random.

Reagent
Final concentration Amount b.Gently flatten the pup's body on the task wipe and firmly stabilize the pup's head on the neck so that the skull is facing up. 4. Identify the injection site at approximately 1 mm lateral from the sagittal suture, located between lambda and bregma.
Note: These are visible through the skin as shown in Figure 1.
a. Insert the needle perpendicular to the surface of the skull at the injection site to a depth of approximately 3 mm.b.Hold the syringe rigidly.c.Slowly inject 1 mL into the ventricle.d.Slowly withdraw the needle.
Note: Make sure the scale is visible to monitor the volume when injecting.
Note: If a reference is needed, mark 3 mm from the tip of the needle with a non-toxic maker.
Note: Do not exceed 2 mL per ventricle as this may lead to ventricular enlargement and cortical thinning.e. Inject the contralateral ventricle using the same procedure.5. Place the pup on the warming pad covered with dry task wipes.
a. Wait until it moves and appears normally.b.Place the pup back into the cage.6. Repeat the procedure until all pups with AAV2/6-CaMKIIa-Cre-ERT2 have been injected.7. Rinse the Hamilton syringe 5 times in 5 Millipore water aliquots each.8. Load 2 mL of AAV2/8-hDlx-Cre-ERT2 into the Hamilton injection syringe and repeat the procedure until all pups have been injected.9. Mark these pups with a laboratory pen so they can be distinguished later.

Note:
Regularly check and remark the pups if necessary.10.When all pups have recovered and returned to the home cage, return the mother to the cage.
a. Ensure that she is attending the pups before returning to the housing facility.
12. At 2 months of age, intraperitoneally inject 1 / 2 of the mice injected with AAV2/6-CaMKIIa-Cre-ERT2 with 1 mg of tamoxifen dissolved in corn oil (10 mg/mL) to induce Cre recombinase activity from ERT2-Cre.a. Inject with tamoxifen for five consecutive days.b.Inject the other 1 / 2 with corn oil alone.
Note: Assign mice receiving tamoxifen at random and use both sexes.
Note: Do not exceed an injection volume of 150 mL as larger volumes can lead to pain, formation of fibrous tissue, perforation of abdominal organs, and hemorrhage.
Note: Tamoxifen dissolution in corn oil is time-consuming.Temperatures >30 C accelerate tamoxifen dissolution in corn oil.Aliquot and store the solutions protected from light at À20 C. c. Repeat step 17 for mice injected with AAV2/8-hDlx-Cre-ERT2. 13.Wait for a minimum of 2 weeks after the last tamoxifen injection for efficient Cre recombination.
Note: Depending on the protein of interest to be expressed or the half-life of the protein to be deleted, a longer duration may be necessary and should be evaluated prior.In general, 2 weeks is sufficient to induce complete Cre recombination from ERT2.
Preparing mouse brains for imaging: ACSF perfusion Timing: 3.5 h for perfusion, followed by overnight incubation in sucrose This section describes a transcardiac perfusion method called ''ACSF perfusion'', which prepares mouse brains for immunofluorescence labeling.i. Oxygenate the ACSF solution for at least 30 min by bubbling the solution with a carbogen (95% oxygen, 5% CO2) gas mixture.
Note: For efficient oxygenation, the end of a carbogen line should have a perforated ending, such as a plastic foam insert at the tip.This leads to the production of numerous small gas bubbles, rather than a few large ones.
ii. Adjust the pH to 7.4 if necessary, then resume oxygenation.15.Assemble the reagents, equipment, and surgical instruments required for perfusion as shown in Figure 2.
a. Keep all solutions on ice.b.Keep ACSF oxygenated.
Note: All equipment and surgical instruments used should be cleaned with 70% ethanol.16.Weigh one animal and anesthetize it with an overdose of pentobarbital.a. Check for depth of anesthesia by pinching the tail and paws.

ACSF Surgical instruments
Note: An intraperitoneal dose of pentobarbital of 50 mg/kg is commonly used.17.Perfuse the mouse transcardially with ACSF.a. Pin the deeply anesthetized animal on a cork board as shown in Figure 3A.b.Open the thorax to expose the heart and major vessels as shown in Figure 3B.c.Hold the heart with a pair of forceps.d.Insert a needle (15-gauge) through the left ventricle into the ascending aorta (follow the axis of the heart).e. Open the right atrium with a sharp hook as shown in Figures 3C and 3D.f.Start the peristaltic pump at a flow rate of up to 16 mL/min (for adult animals).g.When the liver turns beige-yellow, the perfusion is finished (usually less than 2 min).h.Stop the pump.
Note: A good perfusion depends critically on the speed of the surgery.The pump should be started within 1-2 min of opening the thorax.
18. Remove the mouse from the corkboard, decapitate it, and remove the brain using forceps.
a. Cut the brain into multiple pieces depending on the region of interest (sagittal or coronal) as shown in Figures 4A and 4B.
Note: Be careful to not cut through the brain regions of interest, i.e. olfactory bulb/prefrontal cortex or hippocampal formation.
b. Place the pieces into a falcon tube containing 15 mL of 4% PFA fixative as shown in Figure 4C.Note the time.
Note: Do not post-fix the entire brain as this will result in insufficient penetration of the fixative.
19.After 90-120 min of post-PFA fixation, rinse the brain in cold PBS and transfer it to a falcon tube containing 30% sucrose in PBS at 4 C. Keep the post-fixation time consistent for all animals.a. Leave in the fridge until the brain has sunken to the bottom of the tube for cryoprotection (usually overnight).
Note: To distinguish brains from tamoxifen-injected or vehicle-injected mice, mark either of them by sticking a needle through a part of the brain that is of no interest to you.
Note: Do not store at 4 C for longer than 3 days due to the risk of contamination.
Note: For long-term storage, snap-freeze the brains using liquid nitrogen and store them in air-tight containers at À80 C.

Brain sectioning
Timing: 2 h In this step, perfused and cryoprotected mouse brains are sectioned in preparation for immunofluorescence labeling.We used a sliding microtome to obtain free-floating sections, but sections can also be obtained using a cryostat and directly mounted on coated glass slides.Since the brain tissue has only been weakly fixed, the resulting brain sections are relatively fragile.It takes practice to handle these sections during cutting and staining without damaging them.
20. Set up the sliding microtome for brain sectioning.a. Pick sections one by one with a wet brush and collect them in a multi-well plastic box filled with cold PBS (place the box on ice, using a dark background).b.Store the sections at 4 in PBS for up to 24 h.Note: Sections can be mounted directly onto coated glass slides.
Note: For long-term storage at À20 C transfer the sections into a multi-well plastic box filled with anti-freeze solution and cover with parafilm.

Timing: 2 days
This step involves the immunofluorescent labeling of free-floating brain sections to be subsequently visualized by confocal microscopy.Compared to the commonly used immunofluorescent labeling of fixed sections, the free-floating tissue approach allows for better antibody penetration especially when thicker sections are used for 3D reconstruction of the tissue/synapses.22. Fill up a 12-well cell culture plate with Tris-Triton and label wells.
a. Use a wet brush to transfer the brain sections of interest to their corresponding wells as shown in Figure 5. b.Wash for 10 min on a shaker (approximately 50-100 rpm).c.Transfer sections to the second row and wash for 10 min on a shaker.d.Transfer sections to the third row and wash for 10 min on a shaker.
Note: Take a minimum of 3 sections per mouse brain.
Note: Mix brain sections during washing and staining that are intended for comparative analysis to control for the methodology.e. Prepare the primary antibody solution in Tris-Triton solution containing 2% serum (the same species as your secondary antibodies) and 0.2% Triton X-100.
Antibody Mix example:

Note:
The chosen secondary antibodies should be cross-adsorbed against all other species from which primary antibodies are used.Otherwise, the secondary antibodies may cross-react with incorrect primary antibodies and create false signals.In our experience, secondary antibodies from Jackson ImmunoResearch (Alexa, Cyanine, Brilliant Violet dyes) and Sigma Aldrich (CF dyes) work reliably.
Note: In our experience, goat/donkey serum and secondary antibodies can be used interchangeably, as long as no primary antibody is raised in these species.
Note: Labeling for Cre protein in a separate staining is necessary to confirm the neuron subtype-specific transduction of AAVs.
23. Aliquot the primary antibody solution into a labeled 9-well glass plate.

Note:
The maximum number of brain sections per well is usually 5-6.

Note:
The volume per well is usually 500 mL.

Timing: 4-6 h
In this step, stained brain sections are imaged for analysis of synaptic puncta.ACSF perfusion followed by a short post-fixation period allows for the preservation and detection of many synaptic epitopes, which are typically damaged during PFA perfusion.
30.We performed super-resolution microscopy using an Airyscan microscope (CLSM 900 Airyscan, Carl Zeiss).Images were taken using a 63x objective to acquire near-2D slices for synaptic puncta quantification, rather than 3D imaging for the reconstruction of entire tissue blocks.The example images for the synaptic imaging are sum projections of Z-stacks (5 sections, 130 nm sectioning steps) of a 58.8 3 58.8mm region with a pixel size of 35 nm, an optical section thickness of 260 nm, and a 2x scan average with a pixel dwell time of 0.52 ms.
Brain section 9-well glass plate 1°Antibody Solution Note: Choose an appropriate microscope system for the detection of synaptic puncta.Regular confocal systems can be used, but super-resolution systems, such as Airyscan, are preferable.
Note: Keep in mind that when comparing different brain sections from multiple animals, all imaging parameters should be kept constant including tissue focal depth (e.g. 10 mm from the top, which will minimize signal variability due to uneven antibody penetration).
Note: Aim to perform the imaging for all sections within a single session to avoid inconsistent imaging across multiple sessions.
31.Switch on the microscope system and let it run until it has reached a stable temperature.a. Perform any necessary calibrations and other startup procedures to ensure consistent system performance during image acquisition.

Note:
The minimum duration between turning the system on and imaging, as well as necessary calibrations, are provided by the manufacturer.
32. Remove the slide to be imaged from the slide carton and let it warm up to room temperature for a few minutes.a. Clean the slide and place it on the microscope.33.If available, use a low-magnification objective and a ubiquitous marker (such as DAPI or NEUN) to locate the brain region to be imaged.a. Switch to a high-magnification objective for imaging.This will typically be an oil immersion objective.
Note: Do not excite the synaptic channels you wish to analyze when using the eyepiece for locating the target region.Exciting these channels unnecessarily may cause photobleaching and falsify the results.
34. Locate a suitable imaging location within the target region to determine the optimal acquisition settings.
Note: This particular location should not be used during the final imaging, as it will experience some bleaching during testing.
a. Follow your system's procedure to set up the optimal image resolution, scanning speed, and different color channels you wish to image.
Note: These settings should ensure that there is no bleed-through between the different channels.Note: For the analysis of near-2D images, large Z-stacks should be avoided.Generally, we recommend either taking a single image, or a Z-stack no larger than 1 mm in depth.

Brain section
Note: For Airyscan imaging, it is recommended to take a Z-stack with at least 5 optical sections with optimal spacing between them, as determined by the software.This will enable the ''3Dprocessing'' option in the Airyscan processing tab, which is significantly better than the regular 2D Airyscan processing.
b. Set the laser intensity and detector gain for each color channel individually.
Note: Ensure that there are no saturated pixels in the final image.
c. Using these settings, perform imaging of your target region.
Note: It may be helpful to take a high number of images (6-10) of the first brain section and analyze them to get an idea of the image-to-image variability within one specimen.The observed variability can then be used to choose a suitable number of images to take for each subsequent section.

EXPECTED OUTCOMES
The expected outcome of delivering genetically engineered AAVs by neonatal intraventricular injection is the widespread expression of transgenes.For visualization, we obtained coronal and sagittal sections from a 3-month-old mouse injected at P0 with AAV8/2-hSyn1-Cre shown in Figure 8 (Top); long-lasting detection of Cre protein across brain regions highlights the efficiency of utilizing neonatal intraventricular injection for AAV delivery.Of note, cortical and hippocampal brain regions tend to exhibit consistently high expression of transgene, while dorsal brain regions such as the cerebellum tend to exhibit a more variable rate of transduction as shown in Figure 8 (Middle).To investigate the function of pre-or post-synaptically localized proteins in the hippocampal CA1 region, we utilized a combination of AAVs: AAV6/2-CaMKIIa-Cre-ERT2 and AAV8/2-hDlx-Cre-ERT2 (AAV8/2-hSyn1-Cre-ERT2 as control).Following Cre recombination at 2 months, this combination enables the >90% of the targeted cells exhibit deletion of our protein of interest, Adamtsl3, in the adult brain and the investigation of presynaptic (hDlx-Cre-ERT2) and postsynaptic (CaMKIIa-Cre-ERT2) Adamtsl3 function at GABAergic synapses at CA1 pyramidal cell somata (Figure 8 Bottom).
The expected outcome of the described ACSF perfusion and immunofluorescence staining protocol is the superior detection of synaptic proteins compared to PFA-perfused sections.For comparative visualization, we also performed the immunofluorescence staining protocol on PFA-perfused brain sections (Perfusion with PBS and 4% PFA before an additional 3-h post-fixation).The resultant images shown in Figures 9 and 10 were taken in the Stratum Pyramidale and Stratum Radiatum as described in the section ''Image Acquisition''.Images obtained from ACSF and PFA perfused sections were subsequently processed as Z-stack sum-projection modality using identical background subtraction and brightness/contrast settings in the FIJI software (ImageJ).Under identical image processing parameters, the superior signal/noise ratio and spatial resolution of synaptic protein markers in ACSF perfused brain sections are highly visible.Effective labeling as shown is crucial for understanding the function of synaptic proteins; in combination with AAV-mediated segregated manipulation of pre-synaptic or post-synaptic neurons, this protocol thus enables the reliable investigation of pre-or post-synaptically localized proteins, as well as the site-specific function of secreted proteins localized in the synaptic cleft.

LIMITATIONS
Our protocol describes the methods for neonatal injection of AAV, ACSF perfusion, and immunofluorescence labeling in the hippocampal CA1 region.AAV selection as well as antibody selection was therefore performed based on the region of interest and should thus not be used to study other brain regions/synapses.While AAV spread to hippocampal, thalamic, and cortical regions is high, spread to more anterior/posterior regions is variable and should be considered before starting the experiment.Another limiting factor is the proportion of mice lost due to lack of maternal care (varies by strain and breeding pair) following neonatal injection (loss of mice to injection is negligible), which is why a large number of pups is needed to meet relevant n numbers for statistical comparisons.

TROUBLESHOOTING Problem 1: Low transduction efficiency
High AAV transduction efficiency is essential to achieve widespread transgene expression.Transduction efficiency is influenced by many factors, including the precision of P0 injection and AAV titer (Step 1).

Potential solution
To achieve a high transduction efficiency, it is of utmost importance to use high AAV titers and correctly insert the needle at the injection site (location and depth).Only when AAVs are injected into the ventricles, they can move freely from the ventricles into the brain and spread.Therefore, you must practice the injection multiple times in advance to ensure that the injection site is hit correctly, that you are maintaining a 90-degree angle to the surface of the skull, and are not moving the needle up while injecting (common).Additionally, keep your arms firmly planted on a steady surface to minimize any unwanted movement.To maintain AAV titer, avoid freeze-thaw cycles by aliquoting AVVs.

Problem 2: Off-target expression or expression in the absence of tamoxifen induction
The usefulness of tamoxifen-inducible transgene expression depends on its faithful expression.Any expression in cell types other than the desired one, as well as the expression in the correct cell population at the wrong time, can confound results (Step 2).

Potential solution
When selecting an AAV, choose one that has been validated, either in-house or in the literature.Even though well-known promoters such as human Synapsin 1 (hSyn1) or CaMKIIa have been used extensively, it is essential to validate their expression.Validation should occur prior to recombination to evaluate tamoxifen-independent transgene expression and post-recombination using immunofluorescent markers for the transgene and desired cell type.Because tamoxifen-independent recombination has been reported, it is additionally important that your control cohort is AAV injected but did not receive tamoxifen.When your AAV shows off-target expression and high expression in the absence of tamoxifen induction, choose a different one.

Problem 3: Low Cre recombination
The effectiveness of tamoxifen injection is essential to obtain reliable transgene recombination (Step 2).

Potential solution
Make sure you are performing the intraperitoneal injections correctly as bleeding at the injection site, peritonitis or injection into the gastrointestinal tract/bladder reduces the uptake of tamoxifen.Additionally, make sure you keep the tamoxifen cool (4 C) for the duration of injections and always keep it in a light-blocking container or wrapped in foil as tamoxifen is highly light-sensitive.

Problem 4: Poor transcardiac perfusion
Proficient perfusion is vital for the subsequent labeling of synaptic markers using immunofluorescence and is usually visible during perfusion by the liver turning yellowish-white and after the perfusion by a white brain (see Step 3).

Potential solution
There are several indicators of a faulty perfusion.Perfusion solution coming out from an animal's nostrils or mouth, for example, indicates that you have inserted the needle incorrectly and that the solution is flowing into the lungs.Practice to properly insert the blunted perfusion needle into the left ventricle by following the axis of the heart.Also, be careful to not enter the needle too deep, as this may lead to the internal wall of the heart rupturing.If you do accidentally puncture the heart, try clamping the puncture while you continue to perfuse.If necessary, remove and reposition the needle to try again.If all attempts are unsuccessful, you may not be able to use the animal for continuous experimentation, which is why practicing perfusion beforehand is very important.
Problem 5: Low detection of immunofluorescent-labeled proteins Reliable immunofluorescence labeling of synaptic markers is essential for subsequent image acquisition and analysis (Steps 4-10).Weak or no immunofluorescent signals can be caused by many factors including antibody incompatibility or permeabilization method.

Potential solution
When you prepare for your immunofluorescence experiments, it is crucial to verify that both primary and secondary antibodies are compatible to avoid cross-reactivity.Although manufacturers often provide recommended dilutions and incubation times, test these in advance.For some antibodies, increased permeabilized may be necessary and should be adjusted if necessary (see Step 34; 0.4% Triton X-100 increases the penetration of antibodies).The primary and secondary incubation times may be additionally adjusted.There is often a balance regarding incubation time between tissue penetration and background signal.It may be useful to incubate secondary antibodies against ubiquitous targets (e.g., NEUN, CAMK2A, or MAP2) overnight at 4 C to enhance penetration depth, and then the secondary antibodies against synaptic markers for 30 min at room temperature to maintain a low background signal.To mitigate excessive background fluorescence, adjust the antibody concentration and enhance the washing steps to 3 3 15-20 min (see Steps 4 and 5).It is also important to incubate secondary antibodies and store samples in a dark environment to prevent the fading of fluorophore signals, which can occur with prolonged light exposure (Steps 6-10).Another important consideration is the selection of bright, photostable dyes for your secondary antibodies.Some dyes are not bright enough for the detection of low-abundance synaptic proteins.In our experience, the following dyes can be used to detect synaptic markers: Brilliant Violet 421, Alexa 488, Cy3, Alexa 568, CF568, Alexa 647, and Cy5.Other dyes may work as well, but any dye combination should be verified in a test staining for signal quality and lack of spectral overlap.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shiva K.Tyagarajan (shiva.tyagarajan@gmail.com).

Technical contact
Technical questions on executing this protocol should be directed to and will be answered by the technical contacts, Teresa Cramer (crame676@mit.edu) and Andrin Abegg (andrin.abegg@pharma.uzh.ch).

Materials availability
All reagents generated in this study are available from the lead contact without restriction.

Figure 1 .
Figure 1.Neonatal intraventricular injection sites are indicated in red and are located between Bregma and Lambda, 1 mm from the sagittal suture 14. Prepare your reagents.a. Thaw 4% PFA fixative (15 mL per mouse) and keep on ice.b.Prepare 30% sucrose in PBS (15 mL per mouse) for cryoprotection and store at 4 C. c. Prepare ACSF from 10x stock solution.

Figure 2 .
Figure 2. Reagents and surgical instruments required for perfusion are shown for 4% PFA fixative, oxygenated ACSF, and surgical equipment All instruments and reagents should be kept on ice.

Figure 3 .
Figure 3. Mouse transcardial perfusion with ACSF (A) The deeply anesthetized animal is pinned on the corkboard.(B) Thorax is opened to expose the heart.(C) Reference for needle placement: 1. Axis of the heart.2. Right atrium.3. Needle placement.(D) Perfused mouse.

Figure 4 .
Figure 4. Fixation of brain tissue post-perfusion (A) Brains are rinsed in PBS, (B) cut in two or three halves, and (C) transferred into a falcon tube containing 4% PFA.
anti-GABA A R a2 subunit 1:2000 Guinea Pig anti-GABA A R g2 subunit 1:2000 (Continued on next page) Protocol Note: 1 Antibody concentrations are often suggested by the manufacturer and should be tested and adjusted before starting experiments.2 antibody concentrations are also suggested by the manufacturer.

Figure 5 .
Figure 5. Wash brain sections using 12-well cell culture plate filled with Tris-Triton for 3 3 10 min by transferring to respective wells below

Figure 6 .
Figure 6.Prepare 1 Antibody in Tris-Triton solution and incubate free-floating sections using a 9-well glass plate

Figure 7 .
Figure 7. Mounted brain sections on gelatin-coated slides

Figure 10 .
Figure 10.Staining for VGAT (green) in the CA1 Stratum pyramidale and Stratum radiatum of sections obtained from ACSF or PFA perfused brains Filter the solution, cool to room temperature, and adjust the pH to 7.4.Prepare 15 mL per mouse you plan to perfuse.Aliquot and store at À20 C. Aliquots should not be thawed more than once.
Use H 2 O to reach a volume of approximately 900 mL, then adjust pH using 1 M NaOH 1 M HCl if pH is < 7.2 and > 7.4 before using additional H 2 O to reach a final volume of 1000 mL.Store at room temperature for up to 6 months.Before use mix well by stirring.