Tris edta ph 9

Immunohistochemistry was performed according to standard protocols with appropriate positive and negative controls. Antibodies used were: anti-B2M (1:200, antigen retrieval with TRIS/EDTA pH9, Dako), anti-PRDX1 (1:200, antigen retrieval with citrate buffer pH6, Abcam, Cambridge, UK) and anti-PPIA (1:800, antigen retrieval with citrate buffer pH6, Abcam).

Tumor from mice were harvested and placed into 10% neutral buffered formalin (Cellpath). mCherry staining and quantification were performed by UCL Pathology. In brief, deparaffinized hydrated tissue sections underwent antigen unmasking in Tris–EDTA pH 9 (Dako) at high pressure in a pressure cooker for 8 min. After washing and quenching, sections were blocked in 2.5% Horse Serum (Vector ImmPRESS Kit) for 20 min at room temperature. Incubation with primary antibody anti-mCherry (Abcam, ab167453, 1 µg/ml) was for 60 min at room temperature, secondary antibody (anti-Rabbit IgG Polymer Detection Kit, MP-7401, Vector Laboratories) for 30 min at room temperature and DAB + substrate/chromagen (Dako) for 5 min at room temperature prior to counterstaining and mounting.
Slides were scanned in the Hamamatsu NanoZoomer S210 Digital slide scanner. The image analysis has been performed on the whole section with the positive cell counting algorithm from QuPath image analysis software.
Fixation, embedding and CD8 staining were performed at the UCL Institute of Neurology, using the Ventana Discovery XT instrument and Ventana DAB Map detection Kit (760-124). For pre-treatment, Ventana CC1 (950-124) was used. The CD8 antibody (Dako M7103) was used at 1:100 dilution for 1 h before Rabbit anti-murine secondary antibody (Dako E0354) was used.

For histological examination, X-gal + glands were embedded in parafin and cut in 5 μm sections and mounted on positively charged slides. Sections from samples were counter stained with nuclear fast red to identify lacZ+ structures. Sections were subsequently cleared in xylenes and rehydrated through ethanol gradients. Antigen retrieval was performed by heating slides in a boiling water bath for 20 min. in Tris-EDTA pH 9.0 (Dako). Using 3% hydrogen peroxide for 15 minutes at room temperature removed endogenous peroxidase activity. Slides were blocked with normal horse serum (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature and then incubated overnight with primary antibodies to smooth muscle actin (Zymed) or progesterone receptor (Dako) at 4°C. Secondary antibody staining was performed using the R.T.U. Vectastain (goat anti-rabbit/mouse) kit (Vector Laboratories). Staining was visualized using the DAB peroxidase substrate kit (Vector Laboratories) per manufacturer's recommendations. Slides were counterstained with Mayer's hematoxylin (Sigma-Aldrich) and negative tissue controls were included in all immunohistochemical analyses.