The immunohistochemical method of Pandurangan et al33 was adopted with some modifications. Paraffin-embedded tissue sections of 5 μm thickness were rehydrated in xylene and then in graded ethanol solutions. Then, the slides were incubated with HistoVT (10×, pH 7.0) (Nacalai Tesque, Tokyo, Japan) antigen retrieval solution for 20 minutes at 90°C and then cooled to room temperature. The slides were then blocked with 5% bovine serum albumin (BSA) in TBS–Tween 20 (TBST) for 2 hours. The sections were then incubated with mouse monoclonal Nrf2 antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA) overnight at 4°C for immunostaining. After washing the slides thrice with TBST, the sections were incubated with the appropriate secondary antibodies in TBST with 5% BSA for 2 hours at room temperature. The sections were then washed with TBST and incubated for 5–10 minutes with a peroxidase stain from a peroxidase stain DAB kit following the instructions provided by the manufacturer (Nacalai Tesque). Counterstaining was performed using hematoxylin (Cell Path, Mochdre, New-town, UK), and the slides were photographed under a light microscope (Nikon ECLIPSE 80i; Nikon Corporation).
Hematoxylin
Manufactured by Nikon
Sourced in United Kingdom
Hematoxylin is a natural dye extracted from the heartwood of the Logwood tree (Haematoxylum campechianum). It is a widely used stain in histology and cytology laboratories for the visualization of cell nuclei. Hematoxylin stains cell nuclei a blue-purple color, providing contrast and enhancing the visibility of cellular structures.
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Lab products found in correlation
Peroxidase stain dab kit, by Nacalai Tesque (2 mentions)
Eclipse 80i, by Nikon (2 mentions)
Caspase 1, by Proteintech (1 mentions)
Gsdmd, by Proteintech (1 mentions)
Nlrp3, by Proteintech (1 mentions)
Txnip, by Proteintech (1 mentions)
Light microscopy, by Nikon (1 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
Serial xylene, by Thermo Fisher Scientific (1 mentions)
Abc detection ihc kit, by Abcam (1 mentions)
Formalin, by Thermo Fisher Scientific (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Optical microscope, by Nikon (1 mentions)
4 protocols using hematoxylin
1
Immunohistochemical Nrf2 Detection Protocol
2
Immunohistochemical Analysis of Key Inflammatory Markers
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The immunohistochemical method of Pandurangan et al. was adopted with some modifications.
20 Paraffin‐embedded tissue sections of 5 µm thickness were rehydrated in xylene and then in graded ethanol solutions. Then, the slides were incubated with HistoVT (10×, pH 7.0) (Nacalai Tesque) antigen retrieval solution for 20 min at 90°C and then cooled to room temperature. The slides were then blocked with 5% bovine serum albumin (BSA) in TBS‐Tween 20 (TBST) for 2 h. The sections were then incubated with primary antibody overnight at 4°C for immunostaining. The primary antibody: KLF4 (1:100; Proteintech, 11880‐1‐AP, China), TXNIP (1:100; Proteintech, 18243‐1‐AP, China), NLRP3 (1:100; Proteintech, 27458‐1‐AP, China), Caspase‐1 (1:100; Proteintech, 22915‐1‐AP, China), GSDMD (1:100; Proteintech, 20770‐1‐AP, China). After washing the slides thrice with TBST, the sections were incubated with the appropriate secondary antibodies in TBST with 5% BSA for 2 h at room temperature. The sections were then washed with TBST and incubated for 5 min with a peroxidase stain from a peroxidase stain DAB kit following the instructions provided by the manufacturer (Nacalai Tesque). Counterstaining was performed using hematoxylin (Cell Path), and the slides were photographed under a light microscope (Nikon ECLIPSE 80i; Nikon Corporation).
20 Paraffin‐embedded tissue sections of 5 µm thickness were rehydrated in xylene and then in graded ethanol solutions. Then, the slides were incubated with HistoVT (10×, pH 7.0) (Nacalai Tesque) antigen retrieval solution for 20 min at 90°C and then cooled to room temperature. The slides were then blocked with 5% bovine serum albumin (BSA) in TBS‐Tween 20 (TBST) for 2 h. The sections were then incubated with primary antibody overnight at 4°C for immunostaining. The primary antibody: KLF4 (1:100; Proteintech, 11880‐1‐AP, China), TXNIP (1:100; Proteintech, 18243‐1‐AP, China), NLRP3 (1:100; Proteintech, 27458‐1‐AP, China), Caspase‐1 (1:100; Proteintech, 22915‐1‐AP, China), GSDMD (1:100; Proteintech, 20770‐1‐AP, China). After washing the slides thrice with TBST, the sections were incubated with the appropriate secondary antibodies in TBST with 5% BSA for 2 h at room temperature. The sections were then washed with TBST and incubated for 5 min with a peroxidase stain from a peroxidase stain DAB kit following the instructions provided by the manufacturer (Nacalai Tesque). Counterstaining was performed using hematoxylin (Cell Path), and the slides were photographed under a light microscope (Nikon ECLIPSE 80i; Nikon Corporation).
3
Histological and Immunohistochemical Analysis of Colon Tissue
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Dissected colon tissues were fixed in formalin (Thermo Fisher Scientific) for 48 h. Then the tissues were embedded in paraffin, sliced (5-μm), dewaxed in serial xylene (Thermo Fisher Scientific) and rehydrated through graded ethanol solutions (Pharmco-Aaper). For H&E staining, the slides are stained with hematoxylin and eosin (SigmaAldrich), and examined with a light microscopy (Nikon Instruments). The histological scores were evaluated by blind observers according to the following measures: crypt architecture, degree of inflammatory cell infiltration, muscle thickening, goblet cell depletion and crypt abscess. The pathological score is the sum of each individual score. For immunohistochemistry, antigen retrieval was performed by heating the sections in 0.01 M citrate buffer (pH 6.0) to 95 °C for 10 min. The slides were incubated with primary antibodies against occludin, proliferating cell nuclear antigen (PCNA) and β-catenin (Cell Signaling Technology) overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated secondary antibodies were then applied to the sections, followed by the chromogen 4-diaminobenzidine staining according to the instructions of HRP/DAB (ABC) Detection IHC Kit (Abcam). Sections were then counterstained with hematoxylin for 1 min and observed under a light microscope (Nikon Instruments).
4
Quantifying UVB-Induced DNA Damage
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