Intellipath flx

Formalin-fixed, paraffin-embedded tissue was pre-treated in a water bath with Target Retrieval Solution (1:10, Dako, Glostrup, Denmark) for 40 min. Primary antibody to CXCR4 (1:200, order number: ab1640) was purchased from Abcam (Cambridge, UK) and primary antibody to CXCL12 (1:50, order number: MAB350) from R&D Systems (Minneapolis, MN, USA). For staining, kit K5001 (Dako, Glostrup, Denmark) and the automated stainer intelliPATH FLX^® (Biocare Medical, Pacheco, CA, USA) were used according to the manufacturer’s instructions. We included tissues known to contain the respective antigens—reactive tonsils—as controls (positive controls). Replacing the primary antibody with normal serum always produced negative results (negative controls). Both negative and positive controls for CXCR4 and CXCL12 are shown in Figure S11a,b. DLBCL specimens were investigated regarding the staining intensities and percentages of the positive stained DLBCL cells according to the following procedure. For determination of the CXCR4 and CXCL12 expression, the whole section was screened for an equal distribution of positive cells. The determination of the percentage was done by calculating the average percentage of cytoplasmic NR4A1 positive cells in at least ten high-power-fields (0.242 mm² each, field diameter: 555.1 µm). Percentages were rounded to 10%.

The scaffolds were put on glass holders and fixed with an alcoholic fixation solution to prepare them for Ki-67 immunostaining. After rehydration, antigen retrieval was done with heat at pH 6 with target retrieval solution (Dako, Germany, Waldenbronn) according to the manufacturer’s instructions. The immunostaining was conducted on an automated immunohistochemistry staining system (intelli Path FLX, Biocare, USA, Ca, Pacheco) with a mouse anti Ki-67 monoclonal antibody (Zytomed, mouse anti Ki-67 (MSK018) Dako, Germany, Waldenbronn) diluted 1:200 and incubated for 30 minutes at room temperature. For detection the Zytochem-plus HRP kit was used (Zytomed, Germany, Berlin) with incubation times of 20 minutes each from the biotinylated secondary antibody and the Streptavidin conjugate and stained for 12 minutes with a DAB substrate (DAB Substrate Kit, Zytomed, Germany, Berlin) and counterstained with haematoxylin (Dako, Germany, Waldenbronn).
Estradiol and Progesterone concentrations during follicle cultivation with PCL and PCL/gel were evaluated by analyzing removed culture medium during replacement; supernatants from day 2 and day 10 were analyzed in duplicates by using the Access Estradiol and Progesterone Reagent Packs (Beckman Coulter, USA, Ca Brea) with the UniCel DxI 600 Access Immunoassay System (Beckman Coulter, USA, Ca Brea), according to the manufacturer.

Cell lines were fixed in 10% formalin for 1 hour at room temperature. After 1-hour incubation, cells were washed and pelleted in 2% agarose, then paraffin-embedded. The University of Illinois Veterinary Diagnostic Laboratory database was reviewed for cases of iUC among female dogs (for undisputed iUC confirmation) and carcinomas of the prostate. In total, 12 iUC cases and 10 carcinomas of the prostate were identified to be recut for PSMA immunohistochemistry (IHC). IHC staining was performed using an autostainer (intelliPATH FLX, Biocare, Concord, CA). Samples were deparaffinize in xylene and rehydrated in alcohol. Samples were treated with Diva Decloaker and then blocked with Peroxidazed 1 for 5 minutes. A secondary block with Background Punisher was performed for 10 minutes. Samples were incubated with a mouse monoclonal, anti-human PSMA antibody (Novus Biologicals, NBP1–45057) at 1:1000 dilution for 30 minutes. Samples were washed, incubated with a biotinylated secondary antibody for 30 minutes, washed, then incubated with the chromogen IP FLX DAB. Slides were counterstained with hematoxylin. Well characterized human cell lines served as strong positive (LNCaP) and negative/weak positive (PC-3) PSMA controls.

After completing the ex vivo imaging each pancreas was immersed in 10% buffered formalin, fixed for 24–48 h, and routinely processed and embedded in paraffin. Four-micron thick sections were generated at 50-micron intervals through the paraffin block until the entire tissue block was exhausted, resulting in 11–20 separate levels depending upon the size of the pancreas. To identify pancreatic β-cells, one section from each 50-micron interval of each pancreas was immunostained using a murine reactive guinea pig anti-insulin antibody (ab7842; Abcam, Inc., Cambridge MA). For the entire study 635 slides were immunostained. Staining was performed on an automated immunostainer (intelliPATH FLX, BioCare Medical, Concord CA). Briefly, after removal of paraffin in xylene and hydrating the sections through graded ethanols, endogenous peroxidase was blocked by incubation in 3.0% H₂O₂. Non-specific binding sites were blocked by incubation with protein blocking solution (Sniper, BioCare) followed by incubation with the anti-insulin antibody (1∶50; 60 min RT). Sections were subsequently incubated with peroxidase-conjugated goat anti-guinea pig IgG F(ab')2 antibody (#106-036-003; 1∶1000; 30 min RT, Jackson ImmunoResearch Labs, Inc., West Grove PA) and 3,3′-diaminobenzidine (DAB) chromogen. Sections were counterstained with hematoxylin.