A method for freeze-fracture and scanning electron microscopy of isolated mitochondria

Graphical abstract


Specifications
MacDonald, Julie A., William H. Fowle, and Dori C. Woods. "New insights on mitochondrial heterogeneity observed in prepared mitochondrial samples following a method for freezefracture and scanning electron microscopy." Micron. [1] Resource availability N/A

Method details
For the methodology provided here, mitochondria were isolated from cryopreserved C57BL/ 6 mouse tissue, however the method is applicable for many specimens of interest, including cultured cell lines or primary tissue samples from patient biopsy, for two examples-simply adjust the mitochondria isolation step as desired (i.e. use Mitochondria isolation kit (Product # 89,874; Pierce, Rockford, IL, USA) for cultured cells). For any experiments using research animals, ensure all protocols are reviewed and approved by the appropriate institutional animal care and use committee.
a Tissues can be used immediately for processing, or b Tissues can be cryopreserved in freezing medium containing fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO), slow frozen, and stored at À80 C until time of use. 2 Dissociate isolated tissue into small ($200 mg) samples to be used for mitochondria isolation, as per the manufacturers protocol, in this case: Mitochondria isolation kit (Product # 89,801; Pierce, Rockford, IL, USA) a Many kits are commercially available for mitochondrial isolation, as well as published protocols. 3 Collect isolated mitochondria from Step 2 in 1.5 mL microcentrifuge tubes, and wash once by resuspension in 1 mL of a chilled (4 C) respiration-compatible buffer. a Our preferred buffer contains 225 mM D-mannitol, 75 mM sucrose 10 mM KCl, 10 mM TrisÀHCl, 5 mM KH 2 PO 4 at pH 7.0. 4 Pellet washed mitochondria from Step 3 were by centrifugation at 12,000xg for five minutes, and carefully removed by pipet aspiration, and 5 Fix pellet in a primary fixative of 2.5% glutaraldehyde and 2.5% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 overnight (16 h) at 4 C. a Use appropriate personal protective equipment when working with fixatives. Many steps should be completed in a chemical fume hood.
Processing for electron microscopy: 1 Using forceps, carefully remove fixed mitochondrial pellet from "Isolation of mitochondria, Step 5 00 and place in a borosilicate glass vial containing 3 mL wash buffer. Incubate at room temperature (22 C) for 10 min. Repeat for two additional washes. a Wash buffer: 0.1 M sodium cacodylate buffer, pH 7.2 2 Remove final wash buffer by pipet aspiration, and post-fix in 1 mL 1% osmium tetroxide in 0.1 M sodium cacodylate buffer, pH 7.2 for two hours at room temperature (22 C), protected from light. 3 Remove post-fixation buffer and wash pellets a total of three times with 3 mL wash buffer for 10 min at room temperature (22 C). 4 Impregnate samples with DMSO by removing final wash buffer and incubating samples with a 25% solution of DMSO in 0.1 M sodium cacodylate buffer, pH 7.2 for 30 min at room temperature (22 C). a During the first DMSO incubation, prepare a suitable workstation for the LN2 freeze fracture. For this study, we used a small aluminum instrument tray fitted over a polyurethane ice bucket, validated for use with liquid nitrogen, as well as stainless steel forceps and razor blades, and an aluminum weighing spatula to use as a hammer. Freeze fracture and sample mounting: 1 Using forceps, carefully remove DMSO impregnated samples from "Processing for electron microscopy, Step 5 00 and place on prepared frozen surface. a Watch for freezing of residual DMSO surrounding sample to anchor sample to metal surface. 2 Firmly grasp one corner of a razor blade with forceps and place at 45 angle over pellet, making contact with the metal surface with one corner of the blade edge. Using a hammer, strike the elevated corner of the razor blade (not supported by the forceps) in a downward motion to fracture the pellet (Fig. 1) a Repeat as needed until desired number of fragments is obtained. Fracture should create a planar surface through the mitochondrial pellet. 3 Carefully and rapidly collect fractured pellet shards and thaw in a fresh solution containing 50% DMSO in 0.1 M sodium cacodylate buffer, pH 7.2 and incubate samples for 30 min at room temperature (22 C) to gently thaw. a Limit manipulation of the samples to reduce risk of damage to fractured surfaces. 4 Repeat "Processing for electron microscopy" Steps 1-3. 5 Remove final wash buffer by micropipette aspiration, and macerate samples in 3 mL of 0.1% osmium tetroxide in 0.1 M sodium cacodylate buffer, pH 7.2 buffer at room temperature (22 C) overnight (16 h). 6 Remove maceration buffer and wash, as described in "Processing for electron microscopy, Step 1. 00 7 Remove final wash buffer and incubate samples for one hour in a 1% tannic acid solution in 0.1 M sodium cacodylate buffer, pH 7.2 at room temperature (22 C). 8 Remove tannic acid solution, and wash as described in "Processing for electron microscopy, Step 1. 00 9 Remove final wash buffer incubate samples in a final post fixation solution containing 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for one hour at room temperature (22 C), protected from light. 10 Remove post fixation solution wash as described in "Processing for electron microscopy, Step 1. 00 11 Remove final wash buffer and dehydrate samples by moving mitochondria fragments through a graded series of ethanol washes, each wash for 10 min at room temperature. a Ethanol dehydration steps: 30%, 50%, 70%, 85%, 95%, and 100%, using molecular grade ethanol diluted in ultrapure water. 12 Proceed to critical point drying of samples, as per standard processing for SEM.
a For example, in brief: i Cover sample in 100% ethanol, transfer into Critical Point Dryer, such as Samdri1-PVT-3D (Tousimis, Rockland, MD, USA). ii Cool the chamber to À10 C iii Fill the chamber with liquid CO2, close system, and incubate for 2 min. iv Purge chamber, and recool, repeating steps ii-iv for 9-10 cycles. v Fill the chamber with liquid CO2 and begin heating chamber to critical point. vi Incubate at critical point for 4 min. vii Begin to bleed the chamber and slowly reduce pressure until sample can be retrieved. 13 Once dried, mount sample fragments onto a one-inch aluminum sample mount using doublesided carbon adhesive tabs and a colloidal graphite solution, as needed, and allowed to air dry. a When mounting, careful attention should be paid to ensure fractured surfaces are readily available for evaluation by SEM. Fig. 2. Example images resulting from method for freeze-fracture and scanning electron microscopy of isolated mitochondria. Two representative images demonstrating capability to finely resolve mitochondria ultrastructure, including outer membrane, inner membrane, and cristae structures. Scale bars indicate 500 nm.
14 Coat mounted samples using a high-resolution 4 nm sputter coating of platinum, and analyzed samples using a field emission scanning electron microscope such as the Hitachi S-4800 (Hitachi Medical Corporation, Tokyo, Japan). 15 See Fig. 2 for example images.