Anti mac2

Tissues were formalin-fixed and paraffin-embedded. Neutrophil (CD11b⁺Ly6G⁺ cells) were stained as described,^{13 ,18} (link) and counted in 3 random fields per section.
For Mac2, F4-80, and Ki67 staining, after blocking with 3% of H₂O₂ for 20 min at room temperature (RT), and horse serum 1:75 in PBS, 20 min RT; sections were incubated overnight at 4°C with anti-MAC2 (Cedarlane, Burlington, Ontario, Canada), -F4-80 (Biorad, Hercules, CA, United States) or -Ki67 (Cell Signaling, Danvers, MA, United States) in PBS. Tissue sections were then washed and incubated with IgG biotinylated antibody for 30 min at RT, washed in PBS and incubated in ABC reagent (Vector Laboratories, Burlingame, CA, United States) for 60 min at RT, PBS washed, incubated with 3,3′-diaminobenzidine as substrate (Sigma); and haematoxylin counterstained. Positive cells were counted in 3 fields per section. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) was performed according to the manufacturer (TMR Red, Roche, Basel, Switzerland).

Anesthetized mice were first perfused with PBS followed by 4% PFA. Brains were isolated and incubated in 4% PFA for overnight and subsequently placed on graded sucrose (10%, 20%, 30%) for 24 h at 4°C each. The brains were embedded with OCT compound and snap-frozen in a mixture of dry ice and isopentane. Next, 12 μm of serial coronal section of the brain was sliced with a cryostat (Leitz Camera) and mounted immediately onto Superfrost (+) slides (Fisherbrand). Immunofluorescent staining was performed by blocking the sections with 5% BSA containing 0.3% Triton X-100 in PBS for 1 h. The following primary antibodies were incubated overnight at 4°C: Iba-1 (1:200; WAKO), Iba-1 (1:200; Millipore), anti-GFAP (1:300; Abcam), anti-Olig2 (1:200; NovusBiologicals), and anti-Mac2 (1:200; Cedarlane) antibodies. Sections were washed three times with PBS and subsequently incubated for 1 h with the following secondary conjugated antibodies at RT: Alexa Fluor 488 (1:200; Invitrogen) and Alexa Fluor 568 (1:200; Invitrogen). Counterstain was performed with DAPI for 30 s and mounted. For floating sections, 30 μm of the coronal section was sliced and immersed in PBS and followed the procedure described above. The presence of myelin was observed using a Black-Gold II staining kit (TR-100-BG; Biosensis) according to the manufacture’s instruction (Millipore).

Toledo xenograft tumors were formalin fixed, paraffin embedded and sectioned, followed by staining with hematoxylin and anti-CD31 (polyclonal, Abcam) or anti-F480 (clone A3-1, Abcam) or anti-Mac-2 (Clone M3/38, Cedarlane) antibodies. Stained slides were scanned and subjected to stain separation using a customized python implementation that converts from color space to optical density using an unmixing color vector matrix for the individual stains. The individual isolated stains were then projected into a new color space using MATLAB, and overlaid on the unmixed CD31 image in ImageJ. The histology and image analysis were performed by the STTARR facility under the University Health Network, Toronto.

After fixation, AT was embedded in paraffin as described previously (22 (link), 29 (link)). At 4°C sections were incubated overnight with primary antibodies anti-Mac-2 (1:1000; Cedarlane; Burlington, ON, Canada; CL8942AP) and anti-PerilipinA (1:200; Abcam; Cambridge, UK; ab3526). Next, fluorochrome-conjugated secondary antibodies were applied for 1 h at room temperature (1:200; Invitrogen; Waltham, MA, USA). For nuclear staining DAPI (1:10,000; Thermo Fischer Scientific; Waltham, MA, USA; 62248) was used. For control stainings, same routines without primary antibodies were applied. A confocal Leica SPE microscope (Leica; Wetzlar, Germany) was used for image acquisition. Quantification of adipocyte size, interstitial macrophages and CLS density were performed semi-automatically with cellSens Software (Olympus; Hamburg, Germany) as described previously (30 (link)). H&E stainings were performed following standard routines (29 (link)).