Bond max automated immunohistochemistry vision biosystem

Gastric cancer TMA slides were stained for GRAMD1B manually. Following deparaffinization and rehydration of the slides, heat mediated antigen retrieval was carried out using citrate buffer pH 6.0 for 20 minutes, followed by quenching of endogenous peroxidase activity using 3% H₂O₂. Anti-GRAMD1B antibody (1:25) was applied overnight at 4°C. Biotinylated anti-rabbit secondary antibody was then applied on the slides for 1 hour at room temperature, followed by Diaminobenzidine (DAB) development and haematoxylin counter-staining for visualization of the nucleus. pSTAT3 (1:25) staining for TMAs was conducted using the Bond Max Automated Immunohistochemistry Vision Biosystem (Leica Microsystems, Germany). The cytoplasmic and nuclear staining was scored separately and verified by a pathologist from Singapore General Hospital. The positive staining was graded into 4 groups: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong) based on intensity of staining, and the scoring was represented as immunoreactive score (IRS), which takes into account both the percentage of stained cells as well as the intensity of staining. Cut off values for positive staining were determined by calculating mean for each group and statistical analysis using PASW Statistics 18 software was carried out.

Immunohistochemical staining was performed as follows: 3-μm-thick sections were prepared from formalin-fixed paraffin-embedded TMA tissue blocks and dried in a 37 °C oven overnight. The sections were placed in a Bond Max Automated Immunohistochemistry Vision Biosystem (Leica Microsystems GmbH, Wetzlar, Germany) according to the following protocol. First, tissues were deparaffinized and pre-treated with the Epitope Retrieval Solution 2 (pH9) at 100 °C for 20 min. After washing steps, peroxidase blocking was carried out for 10 min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH). Tissues were again washed and then incubated for 30 min with the primary antibody diluted (1:50 anti-Rab35, Abcam ab 152138) in Bond Primary Antibody Diluent (AR9352). Subsequently, tissues were incubated with post primary and polymer for a total of 16 min and developed with DAB-Chromogen for 10 min and counterstained with haematoxilin for 5 min.
Human samples were collected with inform consent between 1995 and 2010, unfortunately, without recording their clinico-pathological features, thus impeding the analysis of RAB35 expression levels with relevant tumour properties.

From each formalin fixed paraffin embedded tumor tissue block, ~3 μm‐thick sections were prepared and single‐marker immunohistochemistry (IHC) was performed in a Bond Max Automated Immunohistochemistry Vision Bio‐system (Leica Microsystems GmbH) using standard protocols.²⁰ Briefly, the sequential steps in IHC were: antigen retrieval, addition of primary antibody, application of a secondary antibody that binds the primary antibody, and addition of a detection reagent to localize the primary antibody. The immune markers used were CD20, CD3, CD4, CD8, CD45RA, CD45RO, Granzyme B, and CD73. The detailed process was mentioned in Supplementary Methods.

We used the immunohistochemical peroxidase method to study the expression of CD44 in kidney bioptates. Immunohistochemical staining was performed using BOND-MAX Automated Immunohistochemistry Vision Biosystem (Leica Microsystems GmbH, Wetzlar, Germany) according to the following protocol. Tissue sections were dewaxed with BOND™ Dewax Solution, 100% Alcohol, BOND™ Wash Solution and pre-treated with BOND™ Epitope Retrieval ER2 Solution at 100 °C for 20 min.
After the washing steps, peroxidase blocking was carried out for 5 min using the Detection Kit Peroxide Block (Bond Polymer Refine Detection Kit DS9800 (Leica Microsystems GmbH)). Then, the sections were incubated with primary antibodies for 30 min at room temperature. We used rabbit polyclonal antibody for CD44 (1:200 dilution; BF-9213, Affinity Bioscience, Cincinnati, OH, USA). For the detection of peroxidase activity, DAB-chromogen was used (Bond Polymer Refine Detection Kit DS9800 (Leica Microsystems GmbH)) for 10 min. After the system produced a brown stain, the specimens were washed and immersed in hematoxylin solution for staining nuclei (for 5 min).
CD44 expression in kidney tissue was evaluated, taking into account the number of CD44 positive cells and the intensity of staining, as it was suggested by Remmele and Stegner (Immunoreactive Scale (IRS), shown in Table 5, Figure S1) [29 (link)].