Anti idh1 r132h antibody

For histology of murine gliomas, brains were carefully removed, post-fixed in 4% paraformaldehyde, and embedded in paraffin. Five μm thick sections were dissected using a microtome. IHC analysis for IDH1-R132H was performed on paraffin-embedded sections using the anti-IDH1-R132H antibody (Dianova). The paraffin was removed using xylene, and the sections were rehydrated in graded alcohol. Antigen retrieval was carried out using preheated target retrieval solution (pH 6.0), and the primary antibodies were incubated overnight. Single immunostains were performed using a standard immunoperoxidase protocol (Vectastain Elite ABC kit, PK-6100; Vector Laboratories, Inc., Burlingame, CA, USA) followed by a diaminobenzidine chromogen reaction (Peroxidase substrate kit, DAB, SK-4100; Vector Lab). All sections were counterstained with Mayer’s hematoxylin and visualized using a LEICA MDLB light microscope.

The IDH1 mutational status was determined by immunohistochemistry using an anti-IDH1 R132H antibody (Dianova). We further validated the results by pyrosequencing (Additional file 1: Figure S1D) in all samples using primers published previously on the Pyromark Q96 ID instrument [27 (link)]. We excluded all IDH1-mutated patient samples from further analysis.

Tissue samples were fixed using 4% phosphate buffered formaldehyde and paraffin-embedded according to standard procedures. H&E staining was performed on 4 μm paraffin sections using standard protocols. Immunohistochemistry was applied using an autostainer (Dako) after heat-induced epitope retrieval in citrate buffer. IDH1 mutation was assessed by immunohistochemistry using an anti-IDH1-R132H antibody (1:20, Dianova). Immunohistochemistry was performed on 3 μm paraffin-embedded tissue sections after deparaffinization and heat-induced epitope retrieval in citrate buffer by using the SignalStain Kit by Cell Signaling according to the manufacturer’s instructions. As primary antibody, Anti-PD-L1-antibody (E1LRN by Cell Signaling) was applied to the tissue in a concentration of 1:200 and incubated overnight at 4°C. The next day, after application of SignalStain® Boost Solution and Secondary Antibody Solution, counterstaining with Meyer’s haemalaun solution was performed. The samples were then mounted and analyzed with an Olympus microscope. PD-L1 positive cells were counted in 6 high-fields (40x magnification) per slide and compared to the total number of cells in each field. From this data, the mean percentage of PD-L1 positive cells was calculated.