Liver or tumor tissues were fixed with 10% neutral buffered formalin and embedded in paraffin. Tissue sections were processed to conduct IHC. Briefly, tissue sections were de‐paraffinized with xylene, rehydrated with various grades of ethanol (100%, 95%, 80%, and 70%), unmasked for antigen retrieval with the provided solution (Vector Laboratories Inc., Burlingame, CA), permeabilized with 0.2% Triton X‐100, blocked with serum, and then incubated with BLOXALL reagent (Vector Laboratories Inc., Burlingame, CA) to quench endogenous peroxidase. Subsequently, the sections were incubated in succession with primary antibodies, secondary antibodies, and DAB substrate at the optimized concentration to develop color. The positive cells were counted in 5 randomly selected fields in each slide with ImageJ software (National Institutes of Health, Bethesda, MD). Abs for cleaved caspase 3 Ab (CAT#9964S), cleaved PARP Ab (CAT#94885S), and CD8a Ab (CAT#98941S) were purchased from Cell Signaling Technology (Danvers, USA), and respectively used for marking the apoptosis cells and effector CD8 T cells.
Bloxall reagent
Manufactured by Vector Laboratories
Sourced in United States, Canada
BLOXALL reagent is a blocking and permeabilization solution used in immunohistochemical and immunocytochemical procedures. It is designed to reduce non-specific background staining and enhance the specificity of antigen-antibody interactions.
Automatically generated - may contain errors
Lab products found in correlation
Abs for cleaved caspase 3 ab, by Cell Signaling Technology (2 mentions)
Cd8a ab, by Cell Signaling Technology (2 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
Rabbit anti foxo1, by Cell Signaling Technology (1 mentions)
Rabbit anti pdx1, by Abcam (1 mentions)
Immpress hrp reagent anti rabbit, by Vector Laboratories (1 mentions)
Axioimager microscope, by Zeiss (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Xylene substitute, by Merck Group (1 mentions)
Novared hrp substrate, by Vector Laboratories (1 mentions)
Cleaved parp antibody, by Cell Signaling Technology (1 mentions)
6 protocols using bloxall reagent
1
Analyzing Apoptosis and CD8+ T Cells in Liver and Tumor Tissues
2
Immunohistochemistry of FFPE Tissues
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Formalin-fixed paraffin-embedded (FFPE) blocks were used to make 4 μm tissue sections. To conduct IHC, tissue sections were de-paraffinized with xylene, rehydrated with various grades of ethanol (100%, 95%, 80%, and 70%), antigen unmasked with solution (Vector Laboratories Inc., Burlingame, CA, USA), permeabilized with 0.2% Triton X-100, blocked with serum, then incubated with BLOXALL reagent (Vector Laboratories Inc., Burlingame, CA, USA) to quench endogenous peroxidase. Subsequently, the sections were incubated in succession with primary antibodies at optimized concentration, secondary antibody, and DAB substrate to develop color. The number of positive cells was counted in 5 randomly selected fields in each slide with ImageJ software.
3
Immunohistochemical Analysis of PDX-1 and FOXO-1
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Immunohistochemistry staining was performed by the Pathology Core at The Centre for Phenogenomics. Tissue sections were submitted to heat-induced epitope retrieval with citrate buffer (pH 6.0) or with TRIS-EDTA (pH 9.0) for 7 min, followed by quenching of endogenous peroxidase with Bloxall reagent (Vector). Non-specific antibody binding was blocked with 2.5% normal horse serum (Vector), followed by incubation for 1 h in Rabbit anti-PDX-1 (Abcam, ab47267, 1:400), or Rabbit anti-FOXO-1 (Cell Signaling Technologies #2880, 1:75). After washes, sections were incubated for 30 min with ImmPRESS HRP reagent Anti-Rabbit (Vector) followed by DAB reagent, and counterstained in Mayer’s hematoxylin. Densitometric analysis was performed using Image J.
4
Histological Analysis of Cellular Infiltration
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To analyze the cellular infiltration, 5-μM-thick sections were cut from paraffin-embedded tissue blocks and placed on charged microscope slides for staining. Sections were deparaffinized, rehydrated, and subjected to routine hematoxylin (Sigma) and eosin (Sigma) staining. Representative images were acquired using a Zeiss AxioImager microscope. To determine the phenotype of the cells at the injection site, 5-μM sections from paraffin-embedded tissues were cut, prepared, and blocked as described above. Endogenous peroxidase/phosphatase activity was blocked using Bloxall reagent (Vector Labs). Anti-F4/80 (6640; Abcam) and anti-CD3 (RM-9107; Thermo Scientific) as primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were used. Peroxidase activity was visualized using NovaRed HRP substrate (Vector Labs). Slides were then counterstained with hematoxylin, dehydrated with ethanol, and cleared with xylene substitute (Sigma). Coverslips were mounted with Organo/Limonene mounting medium (Sigma). Representative images were acquired using a Zeiss AxioImager microscope.
5
Immunohistochemical Analysis of Apoptosis and CD8 T cells
6
Immunohistochemistry Protocol for Collagen Staining
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Four μm tissue sections prepared as described for collagen staining were used for IHC. Briefly, tissue sections were de-paraffinized with xylene, rehydrated with various grades of ethanol (100%, 95%, 80%, and 70%), antigen unmasked with solution (Vector Laboratories Inc., Burlingame, CA), permeabilized with 0.2% Triton X-100, blocked with serum, then incubated with BLOXALL reagent (Vector Laboratories Inc., Burlingame, CA) to quench endogenous peroxidases. Subsequently, the sections were incubated in succession with primary antibodies (Supplementary Table 5 ) at optimized concentration, secondary antibodies, and DAB substrate to develop color. Semi-quantification was conducted with a method like that of Sirius red staining, and the percentage of α-SMA stained area was measured by ImageJ software.
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