Protocol to detect smooth muscle actin-alpha and measure oxidative damage in neonatal mouse intestine

Summary This protocol describes how to characterize α-Smooth muscle actin (αSMA) spatiotemporal expression during mouse small intestinal development. Specific tissue fixation preserves αSMA arrangement in low αSMA expressing cells that are conventionally undetectable under αSMA immunofluorescent stain due to inappropriate fixative-caused artificial actin depolymerization. Parallel analysis of αSMA carbonylation allows estimation of oxidative damage in gut muscular lineage. This approach improves the molecular specificity offered by commercialized kits that estimate total protein carbonyl level in cell lysates without protein specificity. For complete details on the use and execution of this protocol, please refer to Hu et al. (2021).


SUMMARY
This protocol describes how to characterize a-Smooth muscle actin (aSMA) spatiotemporal expression during mouse small intestinal development. Specific tissue fixation preserves aSMA arrangement in low aSMA expressing cells that are conventionally undetectable under aSMA immunofluorescent stain due to inappropriate fixative-caused artificial actin depolymerization. Parallel analysis of aSMA carbonylation allows estimation of oxidative damage in gut muscular lineage. This approach improves the molecular specificity offered by commercialized kits that estimate total protein carbonyl level in cell lysates without protein specificity. For complete details on the use and execution of this protocol, please refer to Hu et al. (2021).

Institutional permissions
All experiments adhered to guidelines of the Institutional Animal Care and Use Committee of Cornell University, under the Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare.
Permissions for animal experiment from the relevant institutions. Any experiments on live vertebrates or higher invertebrates must be performed in accordance with relevant institutional and national guidelines and regulations. Permissions for animal experiments from the relevant institutions are required for the following experiments.
Note: The whole mount immunofluorescent stain protocol works well in mouse embryonic day (E) 16.5-18.5 and neonatal postnatal day (P) 0-9 small intestine. Please refer to previously published protocol (Bernier-Latmani and Petrova, 2016;Suh et al., 2018) for whole mount immunofluorescent stain in adult mouse intestine.
a. If collecting samples from embryonic tissues, euthanize the pregnant dam before embryo isolation per institutional IACUC (Institutional Animal Care and Use Committee) protocol.
Recommended: To reduce auto-fluorescent background from blood cells, proceed to step 2 for an alternative dissection protocol with PBS perfusion.
Pause point: Tissues can be stored at À20 C for at least three months.
CRITICAL: It is necessary to expose the interior of the gut tube prior to fixation if the villous structure is of interest. The villous structure will not be preserved if sample is immersed in methanol without exposing the interior and removing the intestinal content. Methanol fixation can be adapted for fixing other organs (Alarcon-Martinez et al., 2018).
CRITICAL: If 3D structure is not of primary interest. Methanol fixed tissues can be prepared for cryoembedding from this point.
4. Flash freeze tissue in liquid nitrogen for protein carbonyl measurement. Store tissues at À80 C until use.
Pause point: Tissues can be stored at À80 C for at least three months.
CRITICAL: For embryonic and neonatal tissues where genotyping ahead of time is not possible, collect a piece of tissue such as tail or limb for genotyping before isolating the gut. Tissues can be stored at À20 C (methanol fixed) or À80 C (flash frozen) for at least three months.

MATERIALS AND EQUIPMENT
Alternatives: We have successfully used the following antibodies as co-staining markers along with aSMA: vascular endothelial cell marker CD31/PECAM-1 (BD Sciences, 553370) (Figure 4A), cell proliferation marker phospho S10 of Histone H3 (Abcam, Ab5176) ( Figure 4B), and lymphatic endothelial cell marker Lyve1 (Abcam, Ab14917) ( Figures 4C and 4D). These antibodies can be replaced by other antibodies of interest, but the compatibility of such antibodies against methanol fixed antigens needs to be tested beforehand.
Alternatives: This protocol uses the Criterion TM cell, blotter, and precast gels for SDS-PAGE. We visualized our proteins with chemiluminescent substrates and a Bio-Rad ChemiDoc MP system. Other equivalent settings should serve the same purpose.
CRITICAL: H 2 O in this protocol should be high quality water such as double distilled, RO or milliQ equivalent.
CRITICAL: Sodium azide is a hazardous reagent, use a chemical fume hood and wear protective gloves and mask when handling the chemical.
Alternatives: Goat serum can be replaced by donkey serum as described previously (Bernier-Latmani and Petrova, 2016).

Reagent Final concentration Amount
Triton-X 100 0.3% 1.5 mL PBS n/a 500 mL Total n/a 500 mL Can be stored at 20 C-25 C for months.
Biotin-Hydrazide homogenization buffer, pH = 5.5 CRITICAL: Methanol is a hazardous reagent, use a chemical fume hood and wear protective gloves and mask when handling the chemical.

STEP-BY-STEP METHOD DETAILS
Part I: Whole mount immunofluorescent detection of aSMA Timing: 2-3 days aSMA proteins are labeled by fluorescent conjugated antibodies in mouse intestine tissue slices in this part.

Note:
The following protocol is an optimization of a previously published protocol in adult mouse intestine (Bernier-Latmani and Petrova, 2016;Suh et al., 2018). The following protocol is recommended for younger tissues that are less mature and have better tissue permeability than older tissues. Representative images of the whole mount immunofluorescent stain are available in Figure 4.
Note: Conjugated aSMA clone 1A4 antibodies are recommended when staining mouse tissue to minimize background signal.
Note: Methanol fixation will quench intrinsic fluorescent signal such as transgenic GFP or tdTomato. However, using anti-GFP or anti-RFP antibodies, respectively, to detect quenched fluorescent proteins can circumvent this inconvenience.
1. Use spring scissors to separate tissue of interest from methanol-fixed samples into smaller pieces for better penetration. Collect into a round end 2 mL Eppendorf tube in PBS. 2. Wash the tissue 3 times with ice-cold PBS for 5 min each.
Optional: If working with intestines collected from mice older than P9: Before transferring to blocking reagent, permeabilize tissues in PBST-100 (0.3% Triton-X-100) for 6 h at 4 C with gentle rocking. Block for 3 h at 4 C with mild agitation. Use enough blocking reagent to completely submerge all tissues to avoid tissue drying.

Reagent
Final concentration Amount

OPEN ACCESS
3. Incubate with anti-aSMA antibody and other primary antibodies of interest at 4 C for 12-16 h with gentle agitation. Primary antibodies are diluted in fresh blocking reagent with optimal dilution titers. We use 1:100-1:200 for conjugated aSMA antibody.

Note:
The antibody-blocking solution should be filtered with 0.22 mm filter before adding to the samples. Avoid light when handling conjugated antibodies.
Pause point: Samples can be left in primary antibodies at 4 C for more than 1 day if needed.
4. (Go to step 7 if using conjugated antibodies and do not need secondary antibody incubation). Wash with ice-cold PBST-100 (0.3% Triton-100) with gentle rocking at 4 C for five hours, change buffer every hour for 5 times.
Note: Some primary antibody solutions can be reused if stored at À20 C. However, we do not recommend reusing antibodies that cannot be refrozen after thawing.
Note: Fluorescent dyes are light sensitive; minimize ambient light exposure when handling fluorescent dye-conjugated antibodies.
5. Incubate with secondary antibody at 4 C with gentle agitation for 12-16 h.
Optional: Can incubate tissues with DAPI (1:1000 dilution in secondary antibody-blocking solution) to visualize cell nuclei if needed.
Note: Avoid prolonged secondary antibody incubation to minimize background caused by non-specific secondary antibody binding.
Note: For tissues thicker than 0.5 cm, we recommend leaving at 4% PFA for two days.
Note: For tissue embedding, slice the intestine with spring scissors and mount in FocusClear TM /Prolong Gold antifade reagent for confocal imaging (Figure 4) (Bernier-Latmani and Petrova, 2016). The final thickness of the embedded tissue should be no thicker than 2-3 layers of villi.
Note: If the goal is to visualize details in muscularis externa, we recommend cutting the intestine into pieces and mount with villi facing downward ( Figure 5). Alternatively, tissues can be embedded and sectioned after whole mount immunofluorescent staining if needed.

Part II: Tissue protein carbonyl measurement Protein extraction
Timing: 3 h Extract total proteins from the intestine for downstream analysis. This is a separate protocol parallel to aSMA immunofluorescent detection described in Part I.  we use protein carbonyl as a marker to quantify oxidative injury. There will always be baseline protein carbonyls in the cell, therefore it is essential to include a control sample versus the experimental group.
a. Retrieve and thaw samples from À80 C on ice for 5-10 min. b. Make homogenization buffer by adding phosphatase/protease inhibitor cocktail to Biotin-Hydrazide homogenization buffer per label description. Add an adequate amount of buffer to the samples. We recommend adding 500 mL to each intestine sample taken from E18.5-P1.5 mouse. Larger tissue may need more buffer. c. Use a tissue grinder pestle to homogenize tissues completely until no visible tissue chunks remain. d. Shear genomic DNA by passing the homogenized samples up and down through a 1 mL syringe with a needle <25 G. Do this 30 times on ice.
Note: Keep the tubes on ice at all times to avoid protein degradation.
11. Spin at 150 g for 10 min at 4 C. 12. Collect supernatant into a new tube and add SDS to make final concentration of 2%.
Note: We recommend making 20% SDS stock solution in homogenization buffer with protease inhibitor beforehand. Add 1:10 v/v of the premade SDS stock solution to the collected supernatant to make final SDS concentration of 2% in the sample tubes.
13. Heat the sample with SDS at 65 C for 5 min. 14. Spin at 18,000 g for 1 h at 4 C. 15. Collect the supernatant into a new tube and measure protein concentration for each sample.
Note: SDS interferes with several protein assays. Most commercial protein assays include literature on detergent compatibility, which should be consulted prior to use. Protein concentration can be measured at this step with a protein assay compatible with 2% SDS, if samples are run undiluted. To circumvent SDS interference, samples can be diluted to a level of SDS within the protein assay compatibility range. Alternatively, the protein concentration can be determined prior to SDS addition (step 12), and the final concentration calculation adjusted for the volume of SDS added. For any protein assay, a buffer only control should be run to assess any signal contributed by buffer components.

Biotin-hydrazide incubation
Timing: 4 h This step labels protein carbonyl with biotin-hydrazide. 16. Take 100-200 mg of protein samples from each tube, top to 400 mL with homogenization buffer with protease inhibitor from step 12. 17. Add 10 mL of EZ-link Hydrazide-biotin (stock solution: 50 mM in DMSO, store at À20 C) to the sample, bringing the total volume of labeling mix to 410 mL. 18. Incubate the labeling mix at 20 C-25 C with gentle agitation for 2 h. 19. Concentrate sample and remove unreacted Hydrazide-biotin and SDS.
a. Add samples (410 mL) to an Amicon Ultra-0.5 mL centrifugal filter, prepped following manufacturer's instructions. b. Spin at 14,000 g for 10 min. c. Discard flowthrough, add 400 mL PBS, then spin at 14,000 g for 10 min. Repeat twice. d. Recover the samples into a clean tube by spinning at 1,000 g for 2 min. The volume of concentrated samples should be around 40 mL. Add another 20 mL PBS to bring sample volume to 60 mL. Measure protein concentration again.
Alternatives: We use Amicon Ultra 0.5 mL DNA/Protein centrifugal filters, UFC501024, Millipore for step 19. Other protein desalting and concentrating columns or methods such as TCA precipitation should be equally effective.
Note: Concentrated samples should be arranged into three aliquots for streptavidin pulldown assay, aSMA immunoprecipitation (IP), and lysate control. We recommend using roughly 45% of the protein samples for Streptavidin pulldown, 45% for aSMA IP assay, and 10% for the lysate control (step 31).

Timing: 1.5 days
This step isolates all Biotin-Hydrazide labeled proteins (total carbonylated proteins) in the lysate.
20. Pre-wash Streptavidin beads and equilibrate in PBS. a. Mix beads gently by pipetting or inversion of bottle until a homogeneous suspension. Use a cut-off pipette tip to transfer 100 mL of resuspend streptavidin beads to a clean 1.7 mL tube.
Note: Do not vortex the beads.
b. Spin at 500 g for 1 min at 20 C-25 C. Discard supernatant. c. Resuspend the beads with 500 mL of PBS (10 volumes of PBS to beads), centrifuge at 500 g for 2 min and discard supernatant. Repeat two more times. d. Resuspend the beads in 50 mL of PBS. e. Aliquot beads into clean tubes. 21. Add an aliquot of protein samples from step 19 to the tubes with streptavidin beads.

Note:
We use approximately 15 mL of resuspended beads from step 20 for 75-100 mg of biotinylated proteins from step 19, making a 50% beads-sample slurry for step 21.
22. Gently mix the samples and streptavidin beads. Keep rocking for 12-16 h at 4 C. 23. Remove unbound proteins.
a. Spin down the beads at 2,000 g for 2 min in 20 C-25 C. Collect supernatant to confirm pulldown efficiency later (see step 32). b. Resuspend beads with 1 mL of 0.01% SDS in PBS, centrifuge at 2,000 g for 2 min. c. Repeat step 23b four more times. Leave 30 mL of wash buffer in the last wash and resuspend the beads by gentle pipetting. Note: The amount of aSMA antibody used for IP immunoprecipitation depends on aSMA abundance in the sample. Please scale up antibody volume if starting with more samples.
26. Pre-wash protein G Sepharose beads before use. a. Take 100 mL of protein G Sepharose beads suspension with a cut-off pipette tip, spin at 150 g at 4 C for 2 min. Discard supernatant. b. Wash with 1 mL PBS, spin down at 150 g at 4 C for 3 times. c. Aliquot protein G beads slurry to each tube, each tube should have 15-25 mL of protein G beads slurry. Make sure each tube gets equal amount of protein G beads.
Note: Do not vortex the beads.
Note: The aSMA antibody clone 1A4 is mouse IgG2a subtype, which would also be compatible with capture by protein A. If a different aSMA antibody is used for the IP, the choice of protein A and/or G beads for antibody capture should be based on the affinity of the antibody IgG subtype for protein A and G. This information is readily available from commercial supplier websites.
27. After 12-16 h incubation is complete (step 25), add biotin-hydrazide labeled protein-aSMA antibody mix to the tubes with protein G beads. The amount of sample mix to protein G beads slurry should be roughly 1:1 (v/v). Rotate at 4 C for 4 h. 28. Spin at 150 g at 4 C for 2 min to collect beads. Save 20 mL supernatant into a separate tube for IP efficiency control. Remove and discard remaining supernatant, containing unbound material. 29. Wash beads with 500 mL PBST-100 (1% Triton-X-100), spin at 150 g at 4 C for 5 times. 30. Elution: a. Add 30 mL of 23 Laemmli sample buffer with 100 mM DTT and boil on a heating block for 5 min. b. Spin at 150 g at 20 C-25 C for 5 min, collect supernatant for SDS-PAGE.

Preparing controls
Timing: 30 min

SDS-PAGE and western blot
Timing: 1.5 days Carbonylated proteins are separated and detected in this step.
33. Remove the gel comb and tape if using precast gels. Equilibrate the gel to 20 C-25 C.
Optional: Researchers can also prepare their own polyacrylamide gels, versus using precast gels. Gel pouring systems (with instructions for preparation) are available from several suppliers (e.g., Bio-Rad min-PROTEAN handcast system).
34. Rinse/flush the wells thoroughly with running buffer before loading the samples. 35. Load samples and ladder accordingly. 36. Run the SDS-PAGE with 110 V for 90 min. 37. While the SDS-PAGE is running, prepare transfer buffer and cool at 4 C. 38. Soak nitrocellulose membrane in transfer buffer for 10 min and mark the orientation of membrane. 39. Complete a wet transfer at 60 V for 2 h.
Note: This protocol uses the Criterion TM cell, blotter, and precast 8%-16% gels for SDS-PAGE. Appropriate running time and voltage for SDS-PAGE and transfer should be adjusted for different percentage gels and/or transfer systems.
Note: To compare the relative signal between samples, samples must be run and transferred on the same gel. Thus, all aSMA or all streptavidin pulldown samples should be run together on a single gel.
40. Make blocking solution during the wait: 5% BSA in TBST or 5% skim milk in TBST.
Optional: Transfer efficiency and sample loading can be visualized with a Ponceau S staining solution. Wash membrane in TBST until the bands are no longer visible prior to continuing with step 41.
41. Incubate the blot in blocking reagent for 1 h at 20 C-25 C with gentle agitation. 42. Wash the membrane with TBST three times for 5 min each at 20 C-25 C. 43. Incubate in primary antibody (dilute in blocking reagent) or streptavidin-HRP (dilute in TBST) for 12-16 h at 4 C with gentle agitation.
Note: In the initial detection, add the corresponding reagent to the blot: ll OPEN ACCESS a. Streptavidin pulldown blot: aSMA Ab (detect carbonylated aSMA). b. aSMA-IP blot: Streptavidin-HRP (detect all carbonylated proteins in the purified aSMA samples). c. Lysate blot: aSMA Ab (detect total aSMA across samples, ideally band intensity at $42 kDa should be comparable between samples to demonstrate equal amount of aSMA expression across samples).
Note: For blots incubated in streptavidin-HRP, skip to step 46.
Note: Suggested antibody working concentrations are available in the manufacturer's instructions, which may need to be adjusted for optimal signal. We used a 1:400 dilution for primary antibody (aSMA, A2547) in this step, followed with a 1:2000 dilution for secondary antibody (rabbit anti-mouse HRP, ab6728) in step 45. Blots were incubated with Streptavidin-HRP (016030084) at a concentration of 0.1 mg/mL.
Pause point: Blot can be left in primary antibody for more than 16 h if needed.
44. Wash the blots with TBST three times for 5 min each at 20 C-25 C. 45. Incubate the streptavidin pulldown blot and lysate blot with secondary antibody (HRP conjugated anti-mouse secondary antibody in TBST or blocking reagent from 32) for 1 h at 20 C-25 C. 46. Wash the blots with TBST three times for 10 min each at 20 C-25 C. 47. Incubate the blots in fresh luminol buffer mix.
Note: Many chemiluminescent western blotting substrates choices are available, and we used the PerkinElmer Western Lightning Plus-ECL substrate. We visualized our proteins with a Bio-Rad ChemiDoc MP system. Proteins can be visualized by exposing the blot to film or any comparable imager capable of chemiluminescence.
48. Incubate the blots in fresh luminol buffer mix for 1 h to saturate and deplete remaining HRP activity. 49. Return to step 42 for the secondary detection.
Note: In the secondary detection, add the corresponding reagents to the blots for ''primary Ab incubation'': a. Streptavidin pulldown blot: Streptavidin-HRP (for total carbonylated proteins comparison across samples). b. aSMA-IP blot: aSMA Ab (ideally band intensity $42 kDa should be comparable between samples to demonstrate comparable aSMA-IP efficiency across samples). c. Lysate blot: Streptavidin-HRP (for total carbonylated proteins comparison across samples, should show similar trend as in the Streptavidin pulldown blot).
Note: The above-described protocol in Part II is an optimized version from previous protocol dealing with cultured cell lysate and crude adipose tissue extract (Grimsrud et al., 2007;Xu et al., 2014). The previous protocol did not involve specific protein purification steps we described in Streptavidin pulldown and aSMA immunoprecipitation.

EXPECTED OUTCOMES
Whole mount IF Clear aSMA signal should be seen in both the muscularis externa and lamina propria. Vascular smooth muscle should also present in the submucosa ( Figure 4D) and mesentery (Figure 7). The intensity of aSMA signal should be the strongest in the muscularis externa, followed by villous lamina propria vascular smooth muscle cells and villous axial smooth muscles, and the weakest in the villous ll OPEN ACCESS STAR Protocols 3, 101524, September 16, 2022 blood-plexus associated smooth muscle ''star cells.'' The CD31-associated aSMA stain in the star cells is expected starting at E16.5. Axial smooth muscles can be seen no earlier than E17.5-18.5, with more axial muscle fibers established in older tissues. There might be different aSMA staining patterns in the lamina propria throughout the small intestine segments as the organ development in the anterior (duodenum) precedes the posterior (ileum) segments. It is important to note that variation in axial smooth muscle developmental timing was observed across different mouse genetic backgrounds (personal observation, data not shown).

Carbonyl assay
A baseline level of protein carbonyl should be detected in all tissues regardless of genotypes. Sharp and clear bands instead of protein smears should present on the blot ( Figure 6B). If the amount of streptavidin/ aSMA was saturating, there should be no detectable carbonylated proteins in the supernatants collected in step 23a, and no aSMA proteins in the supernatants collected from step 28. Quantitative recovery of material is important to reflect the total material in the samples and avoid erroneous conclusions based on differential protein recovery from the various genotypes. Visualization of the lysates (prior to pulldown) will establish effective biotin-hydrazide labeling as well as indicate the relative levels of carbonyl groups and actin across the samples. The presence of actin signal in the streptavidin pulldowns will be indicative of carbonylated (biotin-labeled) actin. Similarly, the presence of streptavidin signal in the actin IP samples will be representative of carbonylated (biotinlabeled) actin.

OPEN ACCESS
Expected experimental outcome from the secondary detection: Streptavidin pulldown blot (stain with streptavidin-HRP): samples with higher oxidative stress should have overall stronger bands. All carbonylated proteins will be detected in this step, specific target proteins being carbonylated will show enriched signal intensity at the corresponding molecular weight.
aSMA-IP blot (stain with aSMA ab): bands of comparable intensities $42 kDa if the expression and IP effectiveness were comparable across samples.
Lysate blot (stain with streptavidin-HRP): this blot should show the same trend as in the streptavidin pulldown blot.

LIMITATIONS
Whole mount aSMA IF This protocol permits detection of trace aSMA protein detection by preserving the depolymerization-prone antigens with methanol fixation. Nevertheless, methanol fixation may impede the detection of certain antigens that require formaldehyde based-fixation. Some downstream assays are not compatible with methanol fixed tissues as well. For example, formaldehyde-based fixation is recommended in TUNEL assay for minimal DNA breakage. For histological details, we recommend doing Formalin-Fixed-Paraffin-Embedded (FFPE) procedure for H&E stain in parallel to this protocol.

Carbonyl assay
Protein carbonyl is an irreversible protein oxidation product generated under extreme oxidative stress. The stability of protein carbonyl makes it a cumulated injury marker, while providing limited information on the source of real-time free radical generation in the tissue.
Previous protocols have described using biotin-HZ to label protein carbonyls in tissue protein extract. However, these methods rely on molecular weight as the only suggestive factor of target protein identity. This current protocol combines immunoreaction with protein purification methods to measure protein carbonylation in a specific target protein more accurately. Nonetheless, the effectiveness of the pulldown or IP assay depends on the abundance of target protein in the object tissue. Proteins with trace expression or those which are minimally carbonylated are sub-optimal for this protocol. In addition, it is ideal to have comparable protein levels between group of samples. Extra caution will be needed if comparing protein carbonylation between samples with variable protein abundance. Lysate control (input) is therefore necessary to ensure the comparison is proportional instead of an outcome of under-or over-expression of the target protein in the experimental group.

TROUBLESHOOTING Problem 1
No star cell or axial muscle detection aside strong aSMA stain in the muscularis externa in younger tissues from step 9.

Potential solution
Make sure the tissue is fixed in ice-cold methanol and has never exposed to formaldehyde-based fixatives.
Failure to remove the intestinal contents and pancreatic tissue often leads to tissue degradation during processing. Make sure to remove intestinal contents and pancreatic tissue while keeping the tissues on ice and/or at 4 C throughout the entire whole mount staining protocol.
Given that the aSMA signal of axial muscle and star cells is quite low in younger tissues when compared to the gut wall, it is sometimes necessary to overexpose the muscularis externa signal in imaging for acquiring appropriate aSMA stain details in the lamina propria.
Older tissue larger than 0.5 cm 3 may have poor Ab penetrance. Slice tissues into smaller pieces or take extra steps to permeabilize the tissue by bringing through sucrose/glycerol gradient (Bernier-Latmani and Petrova, 2016).

Problem 2
Loss of intrinsic fluorescent signals in methanol fixed tissues after intestine isolation step 3.

Potential solution
In some circumstances, tissues from genetic modified animals carrying intrinsic fluorescent protein expression are used to colocalize aSMA expressions. Methanol fixation will quench the intrinsic fluorescent signal, but this can be overcome by immunofluorescent stain on the intrinsic fluorescent protein of interest. For example, anti-RFP antibody followed by secondary antibody incubation can retrieve transgenic tdTomato signals quenched by methanol.

Problem 3
Protein extract supernatant too viscous to remove after centrifugation in step 12.
Potential solution This is likely caused by excessive genomic DNA contamination in the sample tissues. To overcome this, methods described below are recommended to shear genomic DNA: Increase period of fine needle pulling (step 1D). Sonication.

Problem 4
Lack of distinct bands or signal on the blot in step 47.

Potential solution
Increase protease inhibitor concentration to avoid protein degradation during the procedure if seeing no signals in the lysate control. A baseline level of protein carbonyl should be detected in all tissues regardless of genotypes.
An extended interaction with antibody or streptavidin can be used to increase protein capture. Increased volume, time, and stringency of wash buffer can be used to decrease the presence of bands interpreted as non-specific: for example, signal in streptavidin pulldown from non-biotin-hydrazide treated samples and/or bands outside of the predicted molecular weight of actin ($42 kDa) in the actin pulldown.
The dilution of primary and/or secondary antibody can be adjusted if no signal is observed or if signal reflects an excess of what is interpreted as non-specific bands.

Problem 5
Inefficient pulldown or IP.

Potential solution
Protein carbonyls detected in Streptavidin pulldown supernatant in step 47.

OPEN ACCESS
Adjust the ratio of sample to streptavidin beads by reducing sample amount or increasing streptavidin beads amount.
Increase the incubation time for the sample/streptavidin beads mixture.
Smooth muscle actin detected in supernatant from aSMA IP in step 47.
Adjust the ratio of sample to aSMA antibody by reducing sample amount or increasing aSMA antibody amount.
Increase sample/aSMA antibody incubation time.

Problem 6
Saturated signal on the blot (potentially obscuring differential carbonyl content between samples) in step 47.

Potential solution
Load less sample on the gel or take a shorter exposure when visualizing protein.
Decrease starting protein amount to keep the reaction within its linear range for proper comparison.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Natasza A. Kurpios (natasza.kurpios@cornell.edu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate/analyze [datasets/code].