Goat anti rabbit immunoglobulins biotinylated

The tissue samples were fixed in Bouin’s solution. The samples were processed routinely for paraffin-embedded tissue production into the first week after sampling. A series of 4 μm slides were stained and immunohistochemistry was applied following the Avidin Biotin Complex (ABC) technique. Goat anti-Pig IgA Antibody Affinity Purified (Bethyl Laboratories Inc., Montgomery, TX, USA) was used as primary antibody incubated overnight, and a Polyclonal Goat Anti-Rabbit Immunoglobulins/Biotinylated (Dako, Glostrup, Denmark) as secondary. The Vectastain Elite ABC kit (Vector Laboratories Inc., Newark, CA, USA) was applied for 1 h at 20 °C, and positive labeling was detected using 3,3′-diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark). The tissue sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. Once identified, IgA producing cells were counted by images software analysis under a Zeiss Axioskop 40 light microscope (Carl Zeiss AG, Oberkochen, Germany; Figure 1). The count was the average of the cells counted in 10 non-overlapped fields of 10.000 μm².

Tissue was fixed in 10% formalin neutral buffer solution (Wako, Osaka, Japan), embedded in paraffin, cut into 3.5-µm sections and stained with hematoxylin and eosin (HE). Immunohistochemistry (IHC) was performed with primary antibodies—rabbit anti-mouse CD4 antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-mouse CD8a antibody (Cell Signaling Technology), rabbit anti-Ly6G antibody (Abcam, Cambridge, UK) and rat anti-Ly6C antibody (abcam)—and secondary antibodies—goat anti-rabbit immunoglobulins/biotinylated (Dako, Santa Clara, CA, USA) and simple stain MAX-PO (rat) (Nichirei Biosciences, Tokyo, Japan). The adipocytes were observed with 400× HE and counted, and the area was measured using ImageJ and Adiposoft (NIH, Bethesda, MD, USA).