Dab envision system

To detect the PEDV antigen, immunocytochemistry (ICC) was performed as described previously [15 (link)]. Briefly, Vero cells infected with iPEDVPT-P96 showing typical CPE were fixed with 80% ice-cold acetone, air-dried, and incubated with an in-house anti-PEDV N antibody at room temperature (RT) for 1 h. After washing three times with PBS, a polyclonal anti-rabbit/mouse immunoglobulin, EnVision-DAB⁺ system (Dako, Carpinteria, CA, USA), was applied for 1 h at RT. Following three washes with PBS, the cells were incubated with 3, 3′-diaminobenzidine (DAB) chromogen from a peroxidase DAB substrate kit (Dako, Carpinteria, CA, USA) according to the manufacturer’s instructions. Positive signals were visualized under an inverted light microscope (Nikon, Tokyo, Japan).

The dewaxed and rehydrated tissue sections were permeabilized by 5-min incubation with PBS–0.1% Triton X-100 at room temperature.
After washing in PBS, the sections were incubated with blocking buffer (PBS–0.1% Tween 20 + 5% goat serum) in a humidified chamber for 2 h at room temperature. Then, the sections were washed again in PBS and incubated with primary antibody (1:200 dilution in blocking buffer, rabbit anti-caspase-3 antibody, active, C8487, Sigma) overnight in a humidified chamber at 4 °C. The following day, the slides were incubated with 3.7% H2O2 for 15 min in a humidified chamber at room temperature to inhibit endogenous peroxidase. The secondary antibody was horseradish peroxidase-labeled polymer conjugated to goat anti-rabbit antibody (EnVision + System (DAB), DAKO North America, Carpinteria, CA, USA). The immunocomplex was visualized with 3,3-diaminobenzidine (Dako, Glostrup, Denmark). The sections were lightly counterstained with Harris hematoxylin. Negative controls were established by omitting the primary antibody and substituting IgG for the primary antibodies.
Ten non-consecutive sections of each immunostained kidney were analyzed. The images were captured using a Nikon Eclipse E200 microscope connected to a CCD camera and ImageJ (NIH).
Apoptosis was evaluated by counting the number of caspase-3-positive cells in every HPF analyzed (×40).

Formalin-fixed, paraffin-embedded tissue sections were used for histopathological examination. Histopathological images were photographed using an Olympus BX51 microscope equipped with an Olympus UPlan SAPO 40x objective and a JVC KY-F75 digital camera. IHC was performed using a Dako Autostainer with the EnVision+ System-DAB(Dako, Glostup, Denmark), or a BOND-III with the Bond polymer Refine Detection kit (Leica Microsystems, Melbourne, Australia), with antibodies for CD5, CD10, CD20, BCL2, BCL6, MUM1, MYC, CD2, CD7, CD4, CD8, and CD8β (Table 6). DLBCL cases were classified into GC or non-GC phenotypes, according to the Hans algorithm [42 (link)]. For cases positive for non-CD5-T-cell markers by FCM analysis, additional immunostaining of T-cell markers was performed. On IHC analysis, cells were considered positive for T-cell markers when a small population of neoplastic cells was clearly positive. The cutoffs for MYC and BCL2 IHC were ≥ 40% and ≥ 50%, respectively. The presence of EBV was assessed by in situ hybridization with EBV-encoded small RNA. Fluorescence in situ hybridization (FISH) was performed on formalin-fixed, paraffin-embedded sections using three break-apart probes: BCL2, BCL6, and MYC FISH DNA split signal probes (Dako).

Briefly, five micron thick sections were deparaffinized with xylene, followed by gradation washes in 100%, 95%, 80%,70% ethanol before performing heat antigen retrieval in 10 mM Sodium Citrate buffer (pH 6.0) for 20 minutes. Subsequently, sections were treated with peroxidase block (Dako) for ten minutes followed by overnight incubation at 4° C with the following primary antibodies: anti-90K 1:400 (Santa Cruz #374541), After multiple washes with 1X Tris-buffered saline and Tween 20 (TBST) solution, slides were incubated at room temperature for two hours with corresponding species-specific horseradish peroxidase (HRP) conjugated secondary antibody from EnVision+ System (Dako, K500711-2). Signal was visualized by the HRP-DAB reaction. Counterstaining for nuclei was performed using Mayer’s hematoxylin stain followed by graded dehydration and xylene washes. Coverslips were mounted with Permount (Fisher Scientific). Antibody validation is provided on the manufacturers’ website. Diaminobenzidine (DAB), brightfield staining was performed according to standard protocols using DAB EnVision+ System (Dako) on paraffin sections. The images of immunohistochemical staining were acquired by an Olympus microscope.