Benchmark xt automated slide staining system

Formalin-fixed, paraffin-embedded tissues were cut at 5 μm thickness. We used the BenchMark XT automated slide staining system (Ventana Medical Systems Inc.) to optimize antibody dilutions and staining. The antigen retrieval step (basic pH tris base buffer) was performed using the Ventana CC1 solution. Primary antibody incubation of sections was for 1 hour. The Ventana iView DAB detection kit was used as the chromogen, and the slides were counterstained with hematoxylin.
For double-IHC staining, the BenchMark XT automated slide staining system (Ventana Medical Systems Inc.) was used for deparaffinization and antigen retrieval. The antigen retrieval step was performed using the Ventana CC1 solution, which is a basic pH tris base buffer. Tissue was incubated with the antibody cocktail for 1 hour at 37°C. Tissue was then incubated with mouse AP (alkaline phosphatase) + rabbit horseradish peroxidase (HRP) polymer detection kit (Biocare Mach 2 Double stain 1) for 30 min at RT. Tissues were rinsed with tris-buffered saline and incubated with chromogens Betazoid DAB and Warp Red (both Biocare) for 5 min each, respectively. Slides were counterstained with hematoxylin, dehydrated, cleared, and coverslipped. The Aperio VERSA 8 slide scanning system from Leica Biosystems, equipped with a Point Grey Grasshopper3 color camera for bright-field scanning, was used to analyze stained sections.

Tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin, and 5 μm sections were stained with hematoxylin and eosin (H&E) as described previously [52 (link)]. Immunohistochemistry was performed using a rabbit monoclonal anti-CD4 antibody (1:50; Epitomics, Burlingame/CA, USA) and a Benchmark XT automated slide staining system (Ventana, Tucson, AZ, USA) according to the manufacturer’s recommendations.
Microscopic findings were evaluated using a Zeiss Axioplan microscope (Zeiss, Oberkochen, Germany) with Olympus DP26 digital camera and CellSens Entry software (Olympus, Tokyo, Japan). The height of the mucosa was measured in triplicate, and a mean height value was calculated for each specimen. The number of CD4⁺ lymphocytes in the mucosa was counted in three different high power fields (HPF), and a mean value of CD4⁺ lymphocytes per HPF was calculated.