I use an approach similar to Liz, but I generally combine Nissl and IB4 fluorescent staining to determine the different laminae (see picture below). Lamina I is just 1 neuron or 2 small neurons at the upper border of the gray matter, then lamina IIo is down to the IB4 staining and IB4 staining is lamina IIi. Lamina III is usually the same size of lamina II (o+i). This staining can be performed with free floating SC sections in just 30 min using the IB4-Alexa 647 (1/200, Thermo, # I32450) and Nissl-Alexa 500 (1/400, Thermo, # N21480).
1 Like