Materials and Reagents

Adhesion microscope slides (CITOTEST LABWARE MANUFACTURING, catalog number: 80312-3161-16 )
Note: These slides with special treatment process that electrostatically adheres tissue to the glass without the need for adhesives or protein coatings.
Cover slips (CITOTEST LABWARE MANUFACTURING, catalog number: 80342-1130 )
Cell culture dish (100 x 10 mm) (Corning, catalog number: 430167 )
Animal: 4-month-old Sprague Dawley (SD) rat
Chloral hydrate (Sinopharm Chemical Reagent, catalog number: 30037517 )
Sodium chloride (NaCl) (Guangdong Guanghua Sci-Tech, catalog number: 1.01307.040 )
Paraformaldehyde (PFA) (Guangdong Guanghua Sci-Tech, catalog number: 1.17767.014 )
Phosphate-buffered saline (PBS) (Beyotime Biotechnology, catalog number: C0221A )
Triton X-100 (Sigma-Aldrich, catalog number: V900502 )
Gelatin (Sigma-Aldrich, catalog number: G7041 )
Anti-S100 Protein antibody, clone 15E2E2, produced in mouse (S100) (Merck, catalog number: MAB079-1 )
Anti-Neurofilament 200 antibody produced in rabbit (NF) (Sigma-Aldrich, catalog number: N4142 )
Alexa Fluor^® 488 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A-11001 )
Alexa Fluor^® 568 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A-11011 )
TWEEN^® 20 (Sigma-Aldrich, catalog number: P1379 )
4,6-Diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D9542 )
Mounting medium for fluorescence (Vector Laboratories, catalog number: H-1000 )
10% chloral hydrate (see Recipes)
0.9% NaCl (see Recipes)
4% PFA (see Recipes)
0.1% Triton X-100 (see Recipes)
Blocking buffer (see Recipes)
PBST (see Recipes)
1,000x DAPI (see Recipes)

Equipment

Dissecting scissors and forceps (see Figure 1A)
Perfusion pump (Longer, catalog number: BT300-1F )
Spring scissors (66 Vision Tech, catalog number: 54053B ) (see Figure 2A)
Fine forceps (Fine Science Tools, Dumont, model: #5SF, catalog number: 11252-00 , see Figure 2A)
Black wet chamber (Leibusi, catalog number: 340012 )
Note: The wet chamber (Leibusi, catalog number: 340012 ) was made by a local workshop.
Stereomicroscope (Olympus, catalog number: SZ61 )

Procedure

Anaesthetize a rat with an intraperitoneal injection of 10% chloral hydrate (0.3 ml/100 g) (see Recipes).
Note: Animal use was approved by the Southern Medical University Animal Care and Use Committee and the experimental procedure was performed in accordance with the guidelines for the ethical treatment of animals. All efforts were made to minimize animal sacrifice and suffering.
With big scissors and forceps, expose the heart by cutting the thoracic cavity open.
Using a perfusion pump, perfuse the rat with cold 0.9% NaCl (see Recipes) by transcardial perfusion to clear the blood in the body.
Perfuse the rat with cold 4% PFA (see Recipes) to fix the rat till it becomes stiff.
With big scissors and forceps, cut the skin to expose the biceps femoris and the intermuscular septum (see Figure 1B).
With small scissors and forceps, separate the biceps femoris along the intermuscular septum to expose the sciatic nerve (see Figure 1C).

Figure 1. Expose the sciatic nerve. A. Scissors and forceps used to dissect sciatic nerves; B. The biceps femoris (BF) and the intermuscular septum (IS) are shown after removal of the skin. C. Sciatic nerve is exposed after dissecting the biceps femoris. C’. A magnified image of C shows the sciatic nerve before removal of it from the rat.
Use small scissors to cut the nerve at the knee and the sciatic notch, obtain the nerve and store in 4% PFA at 4 °C overnight for post-fixation.
Use spring scissors to cut a nerve segment of 3-5 mm from the fixed nerve, and place the segment in a dish filled with PBS at room temperature (RT) (see Figure 2B).
Use fine forceps to remove the epineurium wrapping the nerve under a stereomicroscope (see Figure 2C).
Note: The epineurium is the outermost layer of dense irregular connective tissue surrounding the nerve. It appears more transparent than the nerve fasciculus after the fixation.
Transfer the epineurium-free nerve onto an adhesion slide immersed in PBS in a dish (see Figure 2D).
Use fine forceps to tease the nerve into parts under the stereomicroscope and carefully isolate the single individual fibers (see Figure 2E).
Note: While teasing the nerve into parts, some fibers will dissociate from the nerve fasciculus individually or in small groups. Then isolate the dissociated fibers from the fasciculus and carefully tease them into single individual fibers without breaking them. Isolated fibers floating in PBS appear white in color and semi-transparent.
Use fine forceps to carefully brush the single individual nerve fibers onto the adhesion microscope slide (see Figure 2F).
Note: Good adhesion of fibers to slides is essential in this step, and make sure the fibers are well adhered to the slides. The whole fiber attaching to the slide without any detachment suggests good adherence.

Figure 2. Step-by-step procedures of teasing individual fibers. A. Spring scissors and forceps used to tease nerve; B. A nerve segment before the removal of epineurium; C. Nerve epineurium (arrow) is half stripped off from the nerve and the epineurium-free nerve fasciculus (arrowhead). D. Nerve fasciculus immersed in PBS on an adhesion slide in a dish under a stereomicroscope; E. Nerve fasciculus with single individual fibers; F. Straight single individual nerve fibers adhering to the slide.
Lift the slide out of PBS and absorb the liquid left on the slide with absorbent paper.
Air-dry the adhering fibers and store at 4 °C overnight for stronger adhesion.
Immuno-stain the teased fibers (steps 16-26) in the following days as soon as possible for further analysis.
Note: Store slides at -80 °C for long-term storage. However, immunofluorescent staining performed in the next day usually gets the most guaranteed results.
Wash the fibers on the slides 3 times (10 min each) with PBS.
Incubate the fibers with 0.1% Triton X-100 (see Recipes) in PBS for 30 min to penetrate the membrane.
Incubate the fibers with blocking buffer (see Recipes) for 1 h at RT.
Incubate the fibers with primary antibodies overnight at 4 °C.
Note: Primary antibodies of anti-S100 (1:400, diluted in blocking buffer) to show Schwann cell cytoplasm) and anti-NF (1:800, diluted in blocking buffer) to show neuron axons in the present protocol.
Wash the fibers 3 times (10 min each) with PBST (see Recipes).
Incubate the fibers with secondary antibodies for 2 h at RT.
Note: Secondary antibodies of Alexa Fluor® 488 goat anti-mouse IgG (H+L) and Alexa Fluor® 568 goat anti-rabbit IgG (H+L) are used in a dilution of 1:400 in PBST.
Wash the fibers 3 times (10 min each) with PBST.
Incubate the fibers with DAPI (see Recipes) of 1 μg/ml in PBS for 5 min at RT.
Wash the fibers with PBST for 5 min.
Mounted the fibers with mounting medium and coverslip.
Capture images with a fluorescent microscope.

Data analysis

Representative immunofluorescent stained images to show the structures of teased nerve fibers in Figure 3.

Figure 3. Representative immunofluorescent stained images to show the structures of teased nerve fibers. A. A single individual nerve fiber teased from the sciatic nerve of 2-week old rats was double stained with S100 (a specific marker expressed in the cytoplasm of Schwan cells) and neurofilament (NF, a specific marker for showing axon). B. A single individual nerve fiber teased from the sciatic nerve of 4-month old rats was immunostained with S100 to show the detailed structures of Cajal bands (CB) and Schmidt-Lanterman incisures (SLI) which are only exist in adult nerve fibers. PNC, perinuclear cytoplasm; NR, node of Ranvier.
Note: Both of images were stained with DAPI to show the nuclei of Schwann cells.

Recipes

10% chloral hydrate
Dissolve 10 g chloral hydrate in 100 ml distilled water and store at 4 °C, protect from light, and use within 1 week after preparation
0.9% NaCl
Dissolve 9 g NaCl in 1,000 ml distilled water, store at 4 °C, and use within 1 week after preparation
4% PFA
Dilute 40 g PFA in 1,000 ml PBS
Stir at 65 °C until complete dissolution, store at 4 °C, and use within 2 months after preparation
0.1% Triton X-100
Dilute 100 μl Triton X-100 into 100 ml PBS, store at RT, and use within 6 months after preparation
PBST
Dilute 500 μl TWEEN^® 20 into 1,000 ml PBS to a solution containing 0.5% TWEEN^® 20, store at RT, and use within 2 months after preparation
Blocking buffer
Dissolve 0.5 g gelatin (5%, w/v) in 10 ml PBS and add 300 μl Triton X-100 (0.3%) to the buffer, store at -20 °C, and use within 6 months after preparation
1,000x 4,6-diamidino-2-phenylindole (DAPI)
Dissolve 5 mg DAPI in 5 ml distilled water to make 1,000x stock solution of 1 mg/ml, store at -20 °C, use within 2 years after preparation, and dilute to 1x with PBS before use

Acknowledgments

This protocol is adapted from the previously published paper (Wen et al., 2017). This work was supported by the National Key Basic Research Program of China (2014CB542202 and 2014CB542205), National Natural Science Foundation of China (30973095, 81371354 & 81571182); Science and Technology Project of Guangzhou (12C32121609) and Natural Science Foundation of Guangdong Province (S2013010014697) to J Guo.

References

Wen, J., Qian, C., Pan, M., Wang, X., Li, Y., Lu, Y., Zhou, Z., Yan, Q., Li, L., Liu, Z., Wu, W. and Guo, J. (2017). Lentivirus-mediated RNA interference targeting RhoA slacks the migration, proliferation, and myelin formation of Schwann cells. Mol Neurobiol 54(2): 1229-1239.