Ab23990

.The right hemisphere was fixed in 4% paraformaldehyde for 24 h and transferred to 30% sucrose. Hemispheres were embedded in Cryo-Gel and frozen on dry ice. Embedded brains were stored at −80°C until processing. Brains were cryo-sectioned at 20 μm thickness and transferred to frosted slides for immunofluorescence staining. Slices were blocked using 10% normal donkey serum and 5% Bovine Serum Albumin, followed by overnight incubation in primary antibodies: 1:1,500 rabbit anti-Iba1 (Ionized calcium binding adaptor molecule one; Wako 019-19741) and 1:1,000 mouse anti-MHCII (major histocompatibility complex II; abcam ab23990). Secondary antibodies were 1:600 Rhodamine-conjugated donkey anti-rabbit Jackson ImmunoResearch Lab; 711-025-152 and 1:1,000 Alexa Fluor 488 donkey anti-mouse (Jackson ImmunoResearch Lab; 715-545-150). Z-stacks (512 × 512 pixels) were captured using confocal microscopy (Nikon) at 0.5 μm steps using a 60X oil objective lens in the CA1, CA3, and DG of each brain section. The number of microglia in each region was counted by an investigator blinded to the genotype. Microglia density (per mm²) was calculated and averaged per hippocampus per mouse. Maximum projection images of the z-stacks are shown for the representative images.

Rats from various time points during experimental arthritis and respective age matched control animals were killed by CO₂ asphyxia. Adrenal glands were collected and subsequently fixed in phosphate buffered saline (PBS) solution containing 3.7% formaline on ice. Organs were dehydrated in PBS containing 20% sucrose and frozen in Tissue Tek (Sakura Finetek, The Netherlands) on liquid nitrogen. Frozen sections (8–10 µm thick) were obtained with a microtome (Leica, Germany), and after blocking sections were incubated with a primary antibody directed against MHC class II (MHCII) (ab23990, abcam, UK) and an Alexa Fluor fluorescent dye labeled secondary antibody (Invitrogen). Cell nuclei were counterstained with 4′,6-Diamidino-2-phenylindole (DAPI, Sigma Aldrich). With the help of a fluorescence microscope (Leica, Germany), the mean density of MHCII positive cells was determined in 17 randomly selected high power fields from at least three adrenal gland sections per animal.
In further histological studies of rat adrenal gland, we tried to detect B lymphocytes (anti-B220, Cat# 17-0460-82, Thermo Fisher Scientific, Dreieich, Germany), T lymphocytes (anti-CD3, Cat# 13-0030-82, Thermo Fisher Scientific), neutrophils (anti-neutrophil elastase, Cat# 21595, Abcam, UK) combined with a respective Alexa Fluor dye labeled secondary antibody (Invitrogen).

Retinal whole mounts and cross-sections were blocked with PBS containing 5% BSA and 0.3% Triton X-100 for 1 h at room temperature and incubated with primary antibodies diluted in PBS containing 5% BSA and 0.1% Triton X-100 overnight at 4°C (rabbit anti-rat RBPMS (RNA-binding protein with multiple splicing), GeneTex, GTX118619, 1 : 500; rabbit anti-rat IBA1, Wako, 019-19741, 1 : 200; mouse anti-rat CD68, Abcam, ab31630, 1 : 200; mouse anti-rat MHC-II (major histocompatibility complex class II), Abcam, ab23990, 1 : 200; mouse anti-rat GFAP (glial fibrillary acidic protein), Cell Signaling Technology, #3670, 1 : 2000; and rabbit anti-rat C3, Abcam, ab200999, 1 : 200). Then the retinal whole mounts and cross-sections were incubated with secondary antibodies labeled with fluorescent dyes (1 : 1000, Jackson ImmunoResearch) for 2 h at room temperature followed by nuclei staining with DAPI (Vector Laboratories). As negative controls, an adjacent series of sections were processed in parallel without the primary antibodies.