Quantitative Histological Analysis of Murine Brain Tumors

8μm coronal sections were used for frozen sections in all histological studies. The following antibodies were used at the stated dilutions: rabbit polyclonal anti-Iba1, 1:100 (Wako Pure Chemicals, Osaka Japan); Rabbit polyclonal anti-RFP, 1:50 (Rockland), rat monoclonal anti-CD31, 1:100 (Histonova), rabbit anti-Ki67, 1:100 (Abcam). Secondary antibodies conjugated to different Alexa-Fluor dyes (488nm, 555nm, 647nm from Invitrogen) at a dilution of 1:500 in PBS/2%BSA were applied. For nuclear counterstaining, DAPI was used (Sigma). For quantification of Iba1⁺, GFP⁺ or RFP⁺ cells, five to ten images (20x) of tumor and peritumoral regions were taken per mouse brain using CD31 or DAPI staining as a reference on an Olympus FV1000 confocal microscope. TUNEL assay (Sigma) was performed as previously described (19 (link)). Full brain coronal sections were created by multiple area tiled scanning using the same microscope. Cell numbers were counted with FIJI and normalized to a 1 mm² area.