Ribo cc

Slides were stained using a Ventana Discovery XT automated system (Ventana Medical System, Tucson, AZ) as per manufacture’s protocol with proprietary reagents. Briefly, slides were deparaffinized on the automated system with EZ Prep solution (Ventana). Heat-induced antigen retrieval method was used in Cell Conditioning 1 (Ventana). The rabbit primary antibody that reacts to USP10 (#ab72486, Abcam, Cambridge, MA) was used at a 1:400 concentration in Dako antibody diluent (Carpenteria, CA) and incubated for 60 min. The Ventana OmniMap anti-rabbit secondary antibody was used for 8 min. The detection system used was the Ventana ChromoMap Kit and slides were then counterstained with Hematoxylin. Slides were then dehydrated and coverslipped as per normal laboratory protocol. Normal kidney was used as control tissue. For anti-HDAC6 staining, heat-induced antigen retrieval method was used in RiboCC (Ventana). The rabbit primary antibody that reacts to HDAC6, (#C0226-1, Assay Biotech, Sunnyvale, CA) was used at a 1:100 concentration in Dako antibody diluent (Carpenteria, CA) and incubated for 32 min. The Ventana UltraMap anti-rabbit Alk phos secondary antibody was used for 12 min. The detection system used was the Ventana ChromoMap Red kit and slides were then counterstained with Hematoxylin. Slides were then dehydrated and coverslipped as per normal laboratory protocol.

For xenograft samples and pancreatic tissue from KPC mice, dissected tissues were fixed immediately after removal in 10% buffered formalin solution for a maximum of 24 h at room temperature before being dehydrated and paraffin-embedded under a vacuum. The tissue sections were deparaffinized with EZ Prep buffer (Ventana Medical Systems, Santa Clara, USA). Antigen retrieval was performed with CC1 buffer (Ventana Medical Systems), and sections were blocked for 30 min with Background Buster solution (Innovex, Lincoln, RI, USA). IHC detection was performed using the Discovery XT processor (Ventana Medical Systems). All tumor tissues were harvested from mice and fixed in 4% PFA overnight. Fixed tissues were dehydrated, embedded in paraffin, and sliced into 3-µm sections. The tissue sections were deparaffinized with EZ Prep buffer, and antigen retrieval was performed with CC1 buffer and heat treatment in citrate buffer at pH 6.0 (Ribo CC, Ventana Medical Systems). Tumor sections were incubated with the indicated primary antibodies and detection was performed with a DAB detection kit (Ventana Medical Systems) according to the manufacturer’s instructions, followed by counterstaining with hematoxylin (Ventana Medical Systems). Images were obtained using Vectra Polaris (PerkinElmer).

Mouse tissue specimens were immunostained by the labeled streptavidin-biotin method using the Ventana Discovery staining system (Ventana Medical Systems, Oro Valley, AZ, USA) with DISCOVERY DAB Map Detection Kit (Roche, Basel, Switzerland). The anti-mouse CD3 monoclonal antibody (1:150 dilution); the anti-mouse CD68 polyclonal antibody (1:100 dilution), anti-mouse CD163 monoclonal antibody (1:500 dilution), and anti-mouse Ki-67 and PCNA monoclonal antibodies (1:200 dilution) used were diluted with DISCOVERY Antibody Diluent (Roche, Switzerland) [21 (link)]. For CD163 samples, cell conditioning 1 (CC1, pH 8.5, Roche) antigen activation was performed for 60 min at 100 °C. For other samples, RiboCC (pH 6.0, Roche, Switzerland) antigen activation was performed for 60 min at 100 °C. The reaction times of the primary antibody were CD3, 60 min and the others, 12 h. The reaction time of the second antibody were CD3, 60 min and the others, 32 min. Counterstaining was performed with hematoxylin (Roche, Switzerland). Negative control specimens were stained without primary antibodies. Images of non-tumor subserosa were captured by Olympus BX-51/DP-72 under 400× magnification, and the number of CD68- and CD163+ cells per high power field (HPF, 0.196 mm²) was counted.

Mice dissected organs were fixed overnight in 4% paraformaldehyde (pH 7.4), washed in PBS, paraffin-embedded and cut (3 μm).
The presence of RH30 cells in the lungs was studied by immune staining using anti-human CXCR4 antibody (R&D, clone 44716, dilution 1:50), visualization system EnVision FLEX+ (mouse, high pH; Agilent) and hematoxilin/eosin counterstained.
For in situ hybridization, antigen retrieval was first performed with low pH buffer (RiboCC, Roche) and protease III (Roche). Slides were then incubated with the Alu positive control probe II (Ventana, Roche 05272041001). Three stringency washes were performed and slides were incubated with rabbit anti-DNP anti serum. For visualization, OmniRabbit system conjugated with horseradish peroxidase was used (Ventana, Roche). Immunohistochemical reaction was developed using silver as chromogen (Silver kit, Ventana, Roche) and nuclei were counterstained with Carazzi's hematoxylin.
All images were captured on a Carl Zeiss Lab.A1 microscope with an AxioCam ERC5S digital camera (Carl Zeiss).