Sr p37

Surgical specimens from HER2+ breast cancer patients were paraffin embedded and 4 μm sections were cut. After deparaffinization with xylene, tissue sections were rehydrated and endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min. After steaming for 20–30 min using an electric pressure cooker (SR-P37, Panasonic, Tokyo, Japan), the sections were blocked with 5% normal goat serum (Abcam), and incubated with both anti-human CD14 (Diluted in 1:50, Clone 7, mouse monoclonal; Leica) and anti-HER2 (Ready-to-Use, Clone 4B5, rabbit monoclonal; Roche) primary antibodies for 1 h. Alexa Fluor 488-conjugated anti-mouse IgG (diluted in 1:200, ab150117; Abcam) and Alexa Fluor 594-conjugated anti-rabbit IgG (diluted in 1:200, ab150084; Abcam) secondary antibodies were used for immunofluorescence staining of CD14 and HER2, respectively. The secondary antibodies were applied for 1 h prior to mounting with Fluoroshield mounting medium with DAPI (Abcam). To evaluate the correlation between HER2-trogocytosis and a pCR, sections were stained using MACH2 double stain 2 (Biocare Medical) for 1 h, followed by Vulcan Fast Red (Biocare Medical) addition for HER2 staining.

The paraffin sections were rehydrated and immersed in 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. Epitope retrieval was performed by heating the sections at 95 °C in Target Retrieval Solution (pH 9, S2375; Dako) for 30 min in an electric pressure cooker (SR-P37; Panasonic, Tokyo, Japan). All incubations after the blocking step were performed at room temperature. The wash buffer was 0.05 M Tris-buffered saline with 0.05% Tween 20 (pH 7.6). Following blocking with 5% normal goat serum (Abcam) in PBS for 10 min, the sections were incubated with anti-FOXP3 antibodies (236 A/E7; Abcam) for 1 h.
Histopathologic assessment of the percentage of TILs and FOXP3 was performed on one representative immunohistochemical section of a tumor using methods recommended by the International TILs Working Group 2014^{18 (link)}. % TILs was defined as the percentage of lymphocyte area per tumor area in vivo samples. Areas of in situ carcinomas, normal lobules, necrosis, hyalinization, and crush artifacts were not included. Histopathologic evaluation of TILs was performed by two breast pathologists who scored each case independently in a blind manner. The mean values of both observers were obtained as the final scores for each case.