Although relatively simple, the Golgi impregnation technique does have some tricky steps and failure to do them properly results in cells that are unfit for analysis. This technique provides rapid, reproducible labeling of Golgi impregnated neurons. And in addition, the small standard error of the mean allows for comparison between experiments.
The most challenging part about this technique is cutting the cryostat sections, because there are an unusual consistency and fixation, and therefore getting them on the slide flat is a bit of a challenge takes practice. First, place a small quantity of tissue medium on a pre-cool cryostat chuck. Mount the brain blocks on cryostat chucks by thawing one side of the block slightly in a gloved hand and placing it on the tissue medium.
Use a freezing spray on the knife and the block. Use either the anti-roll plate or brush to keep the section flat while cutting. Cut 100 micrometer sections on a cryostat at minus 22 degrees Celsius.
Thaw mount, if possible, by using a room temperature slide and quickly oppose it to the section. Alternatively, keep slides in the cryostat, so they are freezing. Transfer the section with cold forceps or a cold paint brush and then thaw mount.
Mount three to four coronal sections onto subsides. Clean the knife with paper wipes between slices. If the knife requires further cleaning, use 100%ethanol and dry before cutting the next slice.
Place slides in racks, spaced far enough to allow solution access to sections. Place sections in distilled water twice for four minutes before placing them into the Golgi impregnation solution for 10 minutes. Separate the cover slips to ensure only one cover slip is placed on the sections.
Cover the glass slides with a generous amount of mounting medium before placing a glass cover slip on the slide. Once cover slipped, dry slides flagged on any non-porous paper for three to five days, moving them slightly, especially after the first day to avoid sticking. Later transfer the slides to slide holders and ideally, dry slides for at least three weeks before examining them.
To analyze dendritic spine density in pyramidal neurons of both the medial prefrontal cortex and the CA1 region of the hippocampus, measure the dendritic length using in image analysis program. Count the spines on the dendrites using a hand counter and record both length and number of spines. For analysis, choose neurons with cell bodies and dendrites while impregnated, and dendrites that are continuous and distinguishable from the adjacent cells.
The figure shows examples of Golgi impregnated cells in the CA1 region of the hippocampus is shown at low and high power. The figure illustrates an experiment in which basal dendritic spine density was increased on pyramidal cells in adolescent male and female rats after environmental enrichment in CA1 and the medial prefrontal cortex. It's challenging to keep the sections cold enough when cutting and we use a great deal of freezing spray in order to do that.
And then subsequently, it is difficult to get the sections onto the slides and have them dry flat. This technique is used to examine neuroanatomical characteristics. It can be used alone or in combination with other neuroanatomical tracing methods.
