Quantifying NPY-Positive Neurons in Rat Hippocampus

After sacrificing the rats (S1 File), brains were carefully removed from the skull, fixated in 4% formaldehyde solution in phosphate buffer and embedded in paraffin (S1 Table). Coronal brain sections, 5 μm thick, were dewaxed, rehydrated and treated with citrate buffer (pH 6.0) in a microwave for antigen retrieval. Endogenous peroxidase activity was blocked with 3% H₂O₂, and nonspecific labeling was blocked by commercial protein block (Novocastra, UK). Slices were incubated in rabbit polyclonal anti-NPY (1:250, AbD Serotec) overnight at room temperature. The immunohistochemistry procedure on formalin-fixed paraffin-embedded tissue was done according to the methodology described by Nowak and coworkers [24 (link)] with incubation in primary antibody overnight, with slight modification. Labeling was performed using biotin-conjugated secondary antibodies, followed by streptavidin-HRP, and visualization was done with 3,3’-diaminobenzidine (DAB) chromogen (all components from Peroxidase Detection System RE 7120-K, Novocastra, UK). Finally, sections were counterstained with Mayer’s hematoxylin and covered. The staining specificity was checked by omitting the primary antiserum. No immunoreactivity was detected in these sections. Image capturing of NPY stained hippocampal slices was done on Leica DM4000 B LED microscope with digital camera Leica DFC295 and by using Leica Application Suite (LAS, v4.4.0) software system. The surface area of each of the hippocampal regions (CA1, CA2/3, DG) in the chosen sections was measured by the above-mentioned software system and the number of NPY immunoreactive cells was counted in each of those areas (S2 Table), after which the number of counted immunoreactive neurons was expressed per 1 mm² of investigated region in order to standardize the number of counted cells. The counting was always done on the dorsal hippocampus (level of section was 3.80 mm caudal to the bregma, according to Paxinos and Watson stereotaxic atlas [25 ]), on one hippocampal section per animal, and on all animals from control and experimental groups (11 rats per group).