Goat anti mouse f ab 2 fragment

Adjacent sections of 5 μm thickness from Formalin Fixed Paraffin Embedded (FFPE) samples were used for immunohistochemical analysis. The sections were incubated with antibodies against P2RY12 (1:100; Sigma, Sweden); CD68 (1:800; Dako, Denmark) GFAP (1:200; Dako, Denmark); CD45 (1:100; Dako, Denmark) and CD163 (1:400; Abd Serotec, USA). The staining procedure and scanning of the stained sections were performed according to the protocol described previously [40 (link)]. For double labeling, Alexa Fluor 488 and 555-conjugated secondary antibodies (1:200, Thermo Fisher Scientific, The Netherlands) were used for detection. For triple staining, Goat anti-Mouse F(ab)2 fragment (Jackson ImmunoResearch, WestGrove, PA, USA) was used to block background epitopes and Alexa Fluor 647-conjugated secondary antibody (Thermo Fisher Scientific, The Netherlands) was used for detection. The fluorescent labeled samples were analyzed by using the confocal microscope LSM 700 (Zeiss, The Netherlands). Signal positive areas and staining intensity were quantified using the Image J program to five high power field (40x) areas of each immunostained slide.

Acini structures were fixed and assessed by indirect immunofluorescence. Briefly, acini were fixed with 2% paraformaldehyde in PBS for 20 min and permeabilised with 0.05% Triton X-100 in acini-PBS (130 mM NaCl, 7 mM Na₂HPO₄, 3.5 mM NaH₂PO₄, pH 8) for 10 min at 4 °C. Acini were washed three times with 100 mM glycine in acini-PBS for 15 min each. Acini were blocked for 1.5 h with primary block solution containing IF buffer (acini-PBS, 7.7 mM NaN₃, 0.1% bovine serum albumin, 0.2% Triton X-100, 0.05% Tween-20) with 10% goat serum (Gibco), then blocked for a further 40 min in secondary block solution (primary block solution with 20 µg/mL goat anti-mouse F(ab)’₂ fragment (Jackson ImmunoResearch, catalog no. 115-006-006)). Primary antibodies were added in primary block solution overnight at 4 °C, then acini were washed three times with IF buffer for 20 min each with gentle rocking. Alexa Fluor® secondary antibodies and DAPI were added in primary block solution for 45 min at room temperature. Acini were washed three times with IF buffer for 20 min each with rocking. Acini were washed once with acini-PBS, then mounted onto cover slips with Fluoromount G (Electron Microscopy Services, catalog no. 1798425). Images were obtained using a Leica SP8 invert confocal laser scanning microscope.