Immunohistochemistry of Microglial Cells in the Rat Hippocampus sectioned with 7000smz

Abstract

(from Effects of psychogenic stress on some peripheral and central inflammatory markers in rats with the different level of excitability of the nervous system, Shalaginova et al, 2021)

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Patients with post-stress pathologies display the signs of inflammation in the peripheral blood as well as in the brain. The mechanisms of such post-stress neuroimmune changes, their contribution to the behavior, the relationship of the intensity of inflammation with genetically determined features have not been clarified. The goal of this work was to evaluate the dynamics of post-stress inflammation in the blood and hippocampus of rats which differ in level of excitability of the nervous system. Rats of two strains (high/low excitability threshold) were subjected to stress according to the K. Hecht protocol and their behavior, neutrophil:lymphocyte ratio and the number of Iba+ cells in the hippocampus were analysed 24 hours, 7 and 24 days after stress exposure. Highly excitable animals show an increase in anxiety-like behavior, in the number of neutrophils compared to lymphocytes as well as in the number of Iba1+ cells in CA1, CA3 and DG areas of the hippocampus in response to stress. Thus, hereditary high excitability of the nervous system is a possible risk factor for the development of post-stress pathologies.

Method

Immunohistochemistry

The brains were dissected in ice-cold phosphate-buffered saline (PBS, pH = 7.4) and fixed in 4% paraformaldehyde (PFA) for immunohistochemistry. Brains were further washed several times in PBS, embedded in 5% agarose (Dia-m, LM) and cut on vibrating microtome 7000 (Campden Instruments LTD, UK) in order to obtain 50 μm serial sections. The agarose surrounding each section was removed before immunohistochemistry stainings. Serial sections were placed in 12-well plates in PBS with 0.1% sodium azide. Each strip of sections was then washed in PBS 3 times for 10 min and incubated in a blocking solution containing 3% donkey serum (Abcam, ab7475) and 0.3% Triton X-100 (Sigma-Aldrich, Lot: 2725C289) overnight. Primary antibodies raised in goat against Iba1 were used in the study (Abcam, ab5076). The sections were incubated with primary antibodies (1:1000) for 48 h, then washed in PBS with 1% donkey serum and subsequently incubated with donkey anti-goat antibodies conjugated to AlexaFluor488 (Abcam, 50129) (1:1000) for 24 h. After the staining all sections were washed 3 times in PBS for 10 min, mounted on glass slides using Fluoroshield mounting medium (Abcam, 104135) and examined under fluorescent microscope Axio imager A2 (Carl Zeiss, Germany) and confocal scanning microscope LSM 780 (Carl Zeiss, Germany) with ZEN software.