Chromomap dab detection kit

Formaldehyde (4%)-fixed specimens were paraffin-embedded and cut at a thickness of 4 μm. Sections were dried for 1 h at 56 °C and immunohistochemical staining was performed with the automated instrument Discovery XT (Ventana medical system, Tucson Arizona, USA) using the Chromomap DAB Detection kit as follows: sections were deparaffinized and rehydrated using EZ prep (Ventana) and washed with Reaction buffer (Ventana). Antigens were retrieved by heating at 90 °C for 30 min in Citrate buffer (pH 6.0; Ribo CC, Ventana) prior to detection with an anti-Ki-67 antibody (ab15580; Abcam, Cambridge, UK).

Consecutive 3-μM sections were cut from the TMA tissue blocks. The expression of Beclin 1, ATG5 and LC3b was analysed immunohistochemically using the following primary antibodies: anti-Beclin 1 (rabbit polyclonal; Novus Biologicals, LLC; no. NB110-87318, dilution 1:200), anti-ATG5 (rabbit polyclonal; Sigma-Aldrich Corporation; no. A0731, dilution 1:1000) and anti-LC3b (rabbit polyclonal; Abcam plc; no. ab48394, dilution 1:200). Beclin 1 and ATG5 stainings were detected by Discovery UltraMap anti-rabbit IgG in combination with ChromoMap DAB Detection Kit (Ventana Medical Systems, Inc.) and LC3b by UltraView Universal DAB Detection Kit (Ventana). After antigen retrieval (microwave oven for 10 minutes at 250 W) immunohistochemistry for Beclin 1 and ATG5 was carried out in a Discovery Ultra stainer (Ventana) and for LC3b in a Benchmark Ultra stainer (Ventana) according to the manufacturer's instructions. Normal prostatic and testicular parenchyma was chosen as positive control. For negative controls, the primary antibody was omitted. The specificity of the commercial antibodies has been thoroughly validated in former studies [31 (link)–34 (link)].

Briefly, tissue samples were sectioned at a thickness of 5 μm. Deparaffinization, rehydration, endogenous peroxidase activity inhibition, and antigen retrieval were all performed on the Ventana Discovery Ultra IHC (Roche Diagnostics) automated stainer. Slides were then incubated with primary antibodies, followed by DISCOVERY HQ and DISCOVERY HQ-HRP system, visualized with ChromoMap DAB detection Kit (Ventana). The slides were then counterstained with haematoxylin (Ventana) and coverslipped. CD4 rabbit monoclonal 1:100, CD8a rabbit monoclonal 1:100, and CD11C rabbit monoclonal 1:100 were all purchased from Cell Signaling and F4/80 rat monoclonal 1:200 from Bio-Rad.

Immunohistochemistry of the FFPE tumor samples was performed on a BenchMark Ultra autostainer (TLE3) or Discovery Ultra autostainer (Glucocorticoid Receptor). Briefly, paraffin sections were cut at 3 µm, heated at 75°C for 28 min and deparaffinized in the instrument with EZ prep solution (Ventana Medical Systems). Heat-induced antigen retrieval was carried out using Cell Conditioning 1 (CC1, Ventana Medical Systems) for 64 min at 95°C.Glucocorticoid Receptor clone D6H2L (Cell Signaling) was detected using 1/600 dilution, 1 hr at 37⁰C and TLE3 using clone CL3573 (1/250 dilution, 1 hr at RT). Bound TLE3 was detected using the OptiView DAB Detection Kit (Ventana Medical Systems). Glucocorticoid Receptor bound antibody was visualized using Anti-Rabbit HQ (Ventana Medical systems) for 12 min at 37⁰C, Anti-HQ HRP (Ventana Medical systems) for 12 min at 37°C, followed by ChromoMap DAB Detection Kit (Ventana Medical Systems). Slides were counterstained with Hematoxylin and Bluing Reagent (Ventana Medical Systems).

Immunohistochemistry (IHC) was performed after Congo red staining. Briefly, tissue sections were deparaffinized in xylene, rehydrated in a graded series of ethanol, and rinsed with distilled water. After antigen retrieval, endogenous peroxidase activity was blocked using H₂O₂ in blocking buffer (1% fetal bovine serum in PBS) for 30 min. Then the slides were incubated with rabbit anti-mouse Iba-1 (Wako, VA, USA) as the primary antibody (Ab) and UltraMap anti-Rb horseradish peroxidase (HRP) (Ventana Medical Systems, Tucson, AZ, USA) as the secondary Ab. The ChromoMap DAB detection kit (Ventana Medical System) was used for detecting the DAB signal and hematoxylin was used for counterstaining. Slides were observed using a light microscope equipped with a color digital camera.

Formaldehyde (4%) fixed specimens were paraffin-embedded and cut at a thickness of 4 μm. Sections were dried for 1 h at 56 °C, and immunohistochemical staining was performed with the automated instrument Discovery XT (Ventana Medical Systems, Tucson, Arizona, USA) using the Chromomap DAB Detection kit as follows: sections were deparaffinized and rehydrated with EZ prep (Ventana, Oro Valley, AZ, USA) and washed with reaction buffer (Ventana, Oro Valley, AZ, USA). The antigens were retrieved with heat treatment in pH 6.0 citrate buffer (Ribo CC, Ventana, Oro Valley, AZ, USA) at 90 °C for 30 min for anti-Ki-67 (ab15580; Abcam, Cambridge, UK), CK-19 (ab52625, Abcam, Cambridge, UK), and α-SMA (ab5694, Abcam, Cambridge, UK).