TITLE: Immunohistochemical labelling of spinal cord sections for chemoarchitectural analysis of segments
AUTHORS: John-Paul Fuller-Jackson, Peregrine B Osborne, Janet R Keast

[DESCRIPTION]
This protocol is used for immunohistochemical visualisation of the chemoarchitecture of the adult rat lumbosacral spinal cord (and other segments for comparison). Spinal cords were sub-dissected into segments, and transverse sections were obtained from across the rostrocaudal axis of each segment. Two combinations of antibodies were used:
Combination 1: neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT) and NeuN
Combination 2: calcitonin gene-related peptide (CGRP), vesicular glutamate transporter 1 (VGlut1) and tyrosine hydroxylase (TH)

[STEPS]
SECTION: Preparation of cryosections
6. Cryoprotect fixed spinal cord segments in PBS containing 30% sucrose. This should be performed at 4 ºC, 24-72h prior to cutting.
SECTION: Preparation of cryosections
7. Embed tissue in cryomold using OCT, freeze in cryostat and cut sections (40 µm), collecting sections progressively across sets of 4 wells to collect 160 µm spaced series.
SECTION: Immunostaining
8. Wash sections in PBS (3 x 10 min)
SECTION: Immunostaining
9. Incubate sections in blocking solution at room temperature for 2 h
SECTION: Immunostaining
10. Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 48-72h. Antibodies are diluted in PBS containing 0.1% sodium azide, 2% horse serum, and 0.5% triton-X.
SECTION: Immunostaining
11. Wash sections in PBS (3 x 10 min)
SECTION: Immunostaining
12. Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) 4 h in the dark. Antibodies are diluted in PBS containing 2% horse serum, and 0.5% triton-X.

SECTION: Immunostaining
13. Wash sections in PBS (3 x 10 min)
SECTION: Immunostaining
14. Mount sections onto glass slides and coverslip in preferred anti-fade mountant.
SECTION: Microscope
15. Labeled neurons are counted and classified according to their immunoreactivity, including only nucleated neuronal profiles in the analysis. 

 

SECTION: Sub-dissection of spinal cord into segments
1. In a silicone gel-lined petri dish, immerse the fixed spinal cord in phosphate-buffered saline (PBS; 0.1 M, pH7.2).
SECTION: Sub-dissection of spinal cord into segments
2. If still present on the spinal cord, carefully remove the dura mater from the outside of the spinal cord using fine forceps and iris scissors. Take care not to damage the spinal cord or remove the spinal roots as these will be needed as landmarks.
SECTION: Sub-dissection of spinal cord into segments
3. Pin the spinal cord flat by laying the spinal cord dorsal surface facing the gel, and individually pinning all of the ventral spinal roots out perpendicularly.
SECTION: Sub-dissection of spinal cord into segments
4. Identify each of the spinal cord segments using the following landmarks:
Each segment is defined by a ventral root, with the boundaries between segments where one set of rootlets ends, and another begins.
In the lumbar spinal cord, the lumbar enlargement is the widest portion, containing segments L3-L5.
The ventral roots of the sacral segments are much thinner than those of the lumbar segments.
SECTION: Sub-dissection of spinal cord into segments
5. Starting with the most caudal segments, use a scalpel blade to sub-dissect each segment, cutting at the exact point between two sets of rootlets. Store the segments in separate tubes of PBS containing 0.1% sodium azide, labelled appropriately until further use.