The overall goal of this procedure is to obtain insight into the anatomical organization as well as expression and localization profiles of specific proteins in peripheral nerve fibers that innervate tissues such as skin and bone. This is accomplished by first performing whole animal perfusion with fixative, followed by dissection or removal of the tissue of interest. Next, the isolated tissues are postfix and the bones are decalcified using a cryostat fine sections of skin and bone tissue are generated and mounted on slides.
The final step of the procedure is to perform single or multiple label immuno staining of these tissue sections with antibodies against proteins that has specifically expressed and localized on peripheral nerve fibers. Ultimately, results can be obtained that show the distribution pattern of specific types of peripheral nerve fibers, as well as the expression and or localization pattern of specific proteins in peripheral nerve fibers through immunofluorescence microscopy using either an epi fluorescence or confocal microscope. This approach can be used to answer key questions about the peripheral nervous system, such as what are the innovation patterns in tissues such as skin and bone?
And this approach can be extended to visceral organs and other tissues. It can also tell us about changes in expression of proteins within these subsets of sensory nerve fibers and the changes that may occur in expression of these proteins. During pathological or developmental processes, Place an animal that has been perfused with 4%PFA onto a cutting mat or other sturdy surface to collect tissue samples from the hind foot.
Hold the foot with the plantar surface facing up and press down firmly with a three millimeter punch biopsy tool positioned in the middle of the foot. Turn the biopsy tool back and forth through 180 degrees to ensure the biopsy tool has cut through the entirety of the foot. Gently remove the biopsy tool from the foot and eject the tissue into a sterile two milliliter tube containing one milliliter of 4%PFA, or 5%PA solution to collect limb bone samples in this case from the femur.
Make a lateral incision along the back of the animal at the level of the pelvis that can continues down along both hind limbs. Cut into the pelvis and surrounding muscle to separate the femur from the pelvis while leaving the proximal head of the femur intact. Next, cut into the tibia or fibula to leave the distal head intact and remove the surrounding muscle and periosteum from the bone shaft.
Place the femur into a two milliliter tube containing one milliliter of 4%PFA or 5%PA solution, and then allow both samples to postfix with gentle mixing on a rocker for 16 to 18 hours at four degrees Celsius. After post fixation, decalcify both samples by transferring them to sterile two milliliter tubes containing 1.5 milliliters of decalcification solution. Allow the plantar punch to rock gently at four degrees Celsius for 16 to 18 hours and the femur for six to seven days.
Change the solution every 24 hours and monitor the bone tissue for loss of rigidity with a pair of forceps following decalcification. Transfer the tissue samples into a sterile two milliliter tube with 1.5 milliliters of Cryoprotectant solution and place the tube back on the rocker for 16 to 18 hours of gentle mixing at four degrees Celsius. To prepare tissue for sectioning, place a bed volume of optimal cutting temperature compound on a cryostat tissue mounting block and allow to freeze in the cryostat chamber at minus 20 degrees Celsius.
Spray the OCT with a cryogenic aerosol to hasten the freezing process if desired. Position the tissue specimen on the bed of OCT for plantar punch tissue. Position the specimens so that uniform transverse sections can be obtained for bone tissue.
Position the specimen to obtain sections running longitudinal to the bone length. Cover both specimens with an additional thin layer of OCT. Then gently spray with cryogenic aerosol until the tissue is frozen.
Place specimen onto the cutting head in the cryostat chamber and allow the tissue specimen block to equilibrate to the cutting temperature for at least one hour to avoid brittle sections. Once the tissue has reached the optimal temperature, begin cutting the specimen to generate 40 micron sections for plantar punch tissue. Collect the cryo sections in 12 well tissue culture plates containing 0.1 molar PB with 10 millimolar sodium azide.
Make sure that the sections remain submerged and store them at four degrees Celsius until ready for immuno staining for bone tissue. Collect the cryo sections directly onto gelatin pre-coded slides, and then allow them to air dry for one hour before storing at minus 20 degrees Celsius. Bone sections on slides stored in this way can be used for immunostaining purposes within two to three months.
Using a razor blade cut six to seven millimeters from the end of a one milliliter micro pipette tip precondition the inside of the tip by aspirating 1%fetal bovine serum in 0.1 molar PB several times to keep the tissue sections from sticking to the walls of the tip. Transfer eight to 10 plantar punch sections to each well of a 24 well tissue culture plate. Then wash them three times with 500 microliters of 0.1 molar PB per well for five minutes with vigorous mixing on a rocker at room temperature.
Next, incubate the sections in blocking solution with gentle mixing for one hour at four degrees Celsius and then continue on with primary and secondary antibody incubations. After a final wash in pb, use a fresh FBS treated trimmed micro pipette to transfer the sections onto slides. Arrange the sections with a paintbrush and absorb any extra moisture avoiding exposure to light.
Incubate the slides at room temperature in order to dry the sections onto the surface for five to 30 minutes. Once dry, apply several drops of mounting medium and slowly. Place a glass cover slip over the sections.
Allow the slides to sit in darkness for five minutes and then seal the cover slip edges with transparent nail polish Before imaging after thawing the slides at room temperature, use a hydrophobic barrier pen to circumscribe the region on the slide containing the bone sections and then air dry for 10 to 15 minutes. Using a pipet. Apply 250 microliters of 0.1 molar PB within the region and wash the bone sections for five minutes.
Drain off the PB by tilting the slide and repeat for two more times. Next, apply 250 microliters of blocking solution and incubate the sections for one hour at four degrees Celsius. After blocking, proceed onto the primary and secondary antibody incubations being sure to keep the slides in a humidified chamber for overnight incubations.
Finally, wash with 500 microliters of 0.05 molar PB for 15 minutes. At room temperature, dip briefly in distilled deionized water to rinse slides and then allow the bone sections to air dry at room temperature for five to 30 minutes avoiding prolonged exposure to light. Apply mounting medium and place a glass cover slip onto sections After letting the slides stand in darkness for five minutes.
Seal the cover slip edges with nail polish using epi fluorescent microscopy. Numerous CGRP and NF 200 positive fibers are seen here as red areas distributed throughout the basement membranes at the epidermal dermal junction in the plantar region, which have been stained anti collagen four in green, both CGRP and NF 200 positive fibers in the plantar region are also visible using confocal microscopy shown again in red as indicated by the arrowheads. While the pan neuronal marker beta three tubulin is expressed widely in the plantar region.
TRP V one staining is mainly confined to small diameter fibers that are also CG RRP positive, both CGRP and NF 200 positive fibers can be detected in limb bone tissue as well shown here distributed throughout the bone matrix in the spongy head region of mouse femur in red using epi fluorescent and confocal microscopy. T RRP V one is also present in limb. Bone tissue shown here in red using confocal microscopy and co localizes with anti CGRP in green.
After watching this video, you should have a good understanding of how to gain insight into the anatomical features of the peripheral sensory innovation in tissues such as skin and bone, as well as the expression and localization of various proteins within these sensory nerve fibers. The entire process from dissection to microscopic analysis should take approximately four days for skin and 11 days for bone tissue.
