Gelatin embedding and LED autofluorescence reduction for rodent spinal cord histology

We present two innovations in histological technique for rodent spinal cord: gelatin embedding and LED photobleaching. Gelatin embedding uses liquid gelatin solution to permeate delicate biological structures then solidify to provide mechanical support throughout dissection, vibratome sectioning, and staining. LED photobleaching uses high-intensity visible light during blocking and primary incubations to reduce autofluorescence in tissue sections before fluorescent secondaries are added. We found gelatin embedding improved mechanical stability without interfering with immunohistochemical staining. Gelatin embedding also preserved some spinal roots and provided an opportunity for dye-less and cut-less tracking of left/right orientation during free-floating staining, which is valuable for tissue samples that have no spare areas that can be marked. LED photobleaching greatly reduced autofluorescence and added essentially no extra time or labor to the process. Descriptions of the techniques and characterization data are provided.