Mca497rt

For immunofluorescence labeling, liver sections were (co-)stained with antibodies directed against GS (1:1000; ab73593, Abcam), SOX9 (1/100; AB5535, Millipore), F4/80 (1:100; MCA497RT, BioRad), HNF4ɑ (1/100; sc6556, Santa Cruz) or CD31 (1:200; MEC13.3, BioLegend). Secondary antibodies were either anti-rabbit AlexaFluor 488; or anti-rabbit, anti-goat or anti-rat AlexaFluor 647 (1:1000, Invitrogen). Hoechst (1:10 000, 62249, Thermo Scientific) was used to counterstain the nuclei. Liver sections were imaged using the Zeiss AxioImager.Z2 microscope.

Tumor tissues were fixed in 4% paraformaldehyde for overnight, and processed for paraffin blocks. Four-micron sections were made and stained by hematoxylin and eosin.
For immunofluorescence staining, 4 μm paraffine sections were deparaffinized, blocked with 3% Donkey serum for 1 hour at r.t., reacted with anti-F4/80 (1/100, Bio-Rad, MCA497RT) overnight at 4 °C, and Donkey anti-Rat IgG (H + L)−488 secondary Ab. Sections were mounted by Antifade Mounting Medium (BECTASHIELD, H-1200).
Histological photos and immunofluorescent photos were taken by IX73 inverted microscope equipped with DP80 digital camera using CellSens software (Olympus). For lower magnification pictures, we used Keyence BZ-X810 unit.

Immunohistochemistry was performed on 5-mm-thick sections mounted on glass slides precoated with 2% silane, as previsouly described^{29 (link)}. The primary antibodies used were: (anti-rabbit polyclonal type IV collagen 1:50 – abcam 6586 [Cambridge, UK); anti-rabbit polyclonal fibronectin 1:50 – abcam 2413; anti-mouse monoclonal TNF-α 1:25 – Santa Cruz Biotechnology sc-52746 [Santa Cruz, CA]; and anti-mouse monoclonal F4-80 1:50 Bio-Rad MCA497RT [Richmond, CA]) diluted in milk at 1%. After this, 35 glomeruli from each rat were obtained using a magnification of X630 on an optical microscope (Leica® – DMLB 100S). The quantitation of renal densities for type IV collagen, fibronectin, TNF-α, and F4/80 positivity in the glomerular area was performed using ImageJ Software® (National Institutes of Health, Bethesda, MD).

Mice were perfused with phosphate-buffered saline (PBS) and tissues were harvested and frozen in optimal control temperature medium (VWR, 95057-838) and stored at − 80 °C until cryosectioning. 20-μm-thick sections were cut using a cryostat followed by immunohistochemistry. For this, EAE cerebellum tissues and MS brain sections were fixed with ice-cold methanol, or with 4% PFA followed by 0.2% Triton X-100, and blocked with 3% BSA before staining with the following primary antibodies: neural/glial antigen 2 (NG2) (Millipore, AB5320, 1:200) and platelet derived growth factor receptor beta (PDGFRβ) (Invitrogen, 16-1420-82, 1:100; R&D Systems, AF385, 1:100), which were used as the primary markers of pericytes. Anti-pan laminin (a kind gift from Dr. L. Sorokin, Westfälische Wilhelms-Universität, Münster, Germany; 1:1000) that stains the basement membranes of post-capillary venules, and anti-CD31, an endothelial marker (Abcam, 28364, 1:50) were used to characterize post-capillary venules and perivascular cuffs. Anti-CD45 (BD Pharmingen, 550539, 1:75) and anti-F4/80 (Biorad, MCA497RT, 1:100) were used for pan-leukocytes and myeloid cells, respectively. Nuclei were visualized with nuclear yellow (Hoechst). Confocal images were acquired in Z-stacks with ‘confocal-in-a-box’ (Olympus Fluoview FV10i confocal microscope) using a 60× oil-immersion objective.