Alexa fluor 594 labeled anti rabbit

Tissue samples were fixed in 4% formalin and paraffin-embedded (FFPE) at the Department of Pathology (University Hospital Tuebingen). The sections were cut briefly in 3 µm sections and stained with Hematoxylin/Eosin and CD61 (clone: 2C9.G3) following standard protocols. For immunofluorescence microscopy, sections were deparaffinized and hydrated in a first step. The heat-induced antigen retrieval method was performed using sodium citrate buffer (pH 6.0) for 30 min. Antigen blocking was performed with Blocking solution (Zytomed) for 60 min. Primary antibodies that were included anti-CD41, mouse, 1:250 (clone: HIP8) and anti-PD-L1, rabbit, 1:200 (clone:28-8). Secondary antibodies include Alexa-Fluor 594 labeled anti-rabbit (1:1000, Invitrogen) and Fluor 488 labeled anti-mouse (1:1000, Invitrogen). DAPI (1:1000, BioLegend) was used for nuclear staining prior mounting the slides with H-1500 Vectashield Hardset. Microscopic analysis was done with an Olympus BX63 microscope and a DP80 camera (Olympus).

Mice were anesthetized and transcardially perfused with 0.9% saline (wt/vol), followed by fresh fixative of 4% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4). Brains were collected and postfixed overnight before coronal sections (50 μm thickness) were taken by using a vibratome (VT1000P; Leica Microsystems, Wetzlar, Germany). Sections were washed with PB and then treated with 1% H₂O₂ (vol/vol) in PB for 20 min to eliminate endogenous peroxidase activity. After washing in PB several times, the sections were preincubated with 0.2% Triton X-100 (Sigma-Aldrich) for 30 min at room temperature (RT) and then incubated with primary antibodies (antibody to rabbit Iba-1, 1 : 1000 dilution for overnight at RT, Wako, Osaka, Japan; antibody to rabbit c-Fos, 1 : 2000 dilution for overnight at RT, Santa Cruz; antibody to sheep α-MSH, 1 : 10,000 dilution for overnight at 4°C, Millipore). Immunofluorescence was performed with secondary antibodies (Alexa Fluor 594-labeled anti-rabbit, 1 : 1000 dilution for 2 h at RT, Invitrogen Life Technologies, Carlsbad, CA, USA; Alexa Fluor 647-labeled anti-sheep, 1 : 500 dilution for 1 h at RT, Life Technologies). For diaminobenzidine- (DAB-) based Iba-1 IHC, sections were extensively washed and incubated with biotinylated secondary antibody to rabbit, ABC reagent (Vector Laboratories, Burlingame, CA, USA), and DAB substrate (Vector Laboratories).