Sections 5 μm thick of the lacrimal gland were deparaffinized, hydrated, processed in antigen retrieval solution (Abcam Inc., Cambridge, UK), and exposed to 3% H2O2 solution (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) for 30 min. For immunohistochemical analysis, the slides were incubated with primary antibodies against cluster of differentiation 4 (CD4; Novus Biologicals, Littleton, CO, USA), interleukin-17 (IL-17; Abcam Inc., Cambridge, UK), and tumor necrosis factor alpha (TNF-α; Abcam Inc., Cambridge, UK) for 1 h. Subsequently, the sections were incubated with secondary antibodies (DAKO Corp, Glostrup, Denmark) for 40 min, followed by probing with diaminobenzidine chromogen, and counterstained with Mayer’s hematoxylin (YD Diagnostics Co., Yongin, Korea). The stained slides were photographed using an imaging system (Thermo Fisher Scientific). The quantitative analysis of histological staining for CD4, IL-17, and TNF-α was performed using the “threshold tool” of ImageJ® (National Institutes of Health, Bethesda, MD, USA).
Antigen retrieval solution
Manufactured by Abcam
Sourced in United Kingdom
Antigen retrieval solution is a laboratory reagent used to unmask or expose target antigens in fixed tissue samples, which is a necessary step in certain immunohistochemical and immunofluorescence techniques. The solution helps to restore the antigenicity of the target proteins that may have been obscured or altered during the fixation process.
Automatically generated - may contain errors
Lab products found in correlation
Rm2125, by Leica (4 mentions)
H2o2 solution, by Merck Group (3 mentions)
Tnf α, by Abcam (2 mentions)
Il 17, by Abcam (2 mentions)
Evos fl auto 2 imaging system, by Thermo Fisher Scientific (2 mentions)
Mayer s hematoxylin, by Merck Group (2 mentions)
Ab205723, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Goat anti rabbit ig hrp, by Southern Biotech (1 mentions)
Anti itga3, by Merck Group (1 mentions)
Hematoxylin, by Leica (1 mentions)
F4 80, by Abcam (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Tnf α antibody, by Abcam (1 mentions)
11 protocols using antigen retrieval solution
1
Immunohistochemical Analysis of Lacrimal Gland
2
Comprehensive Liver Histological Analysis
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The liver tissues were fixed in 4% formaldehyde for two days and embedded in paraffin. The section of blocks was cut into thickness of 4 μm using a microtome (Leica RM2125, Leica Biosystems, Heidelberg, Germany). For hematoxylin and eosin (H&E) staining, the sections were deparaffinized rehydrated, and washed with distilled water to allow the hydrophilic solution to penetrate. After tissue watering, H&E staining was performed to observe liver tissue histological changes. For immunohistochemistry analysis, the sections were deparaffinized, rehydrated, cooked in antigen retrieval solution (Abcam), and dipped in 3% hydrogen peroxide solution for 30 min. Specific primary antibodies were then applied for 1 h at room temperature, and the sections were incubated with secondary antibodies for 40 min. Immunoreactions were visualized with DAB staining, and the sections were counterstained with Mayer’s hematoxylin. The stained sections were dehydrated, mounted and observed using the EVOS FL Auto 2 imaging system (Thermo Fisher Scientific). Densitometric analysis of the data was performed using the ImageJ® software (v1.48, NIH, Bethesda, MD, USA).
3
Immunohistochemical Analysis of Tumor Biomarkers
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Tumor specimens were prepared as described above. Subsequently, the tissue sections were deparaffinized and placed in antigen retrieval solution (Abcam) for 15 min at 100°C. Following incubation in 1% bovine serum albumin (Sigma-Aldrich) for 30 min, primary antibodies [YAP, CD31, cyclinA, cyclinD1, cyclinE; dilution, 1:200] were applied to the slides for 2 h at 37°C. Following washing with PBS, the sections were additionally incubated with the HRP-conjugated donkey anti-goat secondary antibody (dilution, 1:2,000; ab205723; Abcam) and avidin-conjugated horseradish peroxidase (43–4423; Thermo Fisher Scientific, Inc.). Finally, the sections were treated with 3,3′-diaminobenzidine, and counterstained with hematoxylin, and dehydrated. A negative control was performed by replacement of primary antibody with PBS. Two pathologists reviewed the results, with IHC staining intensity determined by Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
4
Integrin A3 Immunohistochemistry in Kidney Biopsies
5
Immunostaining Protocol with Modifications
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Immunostaining was performed as previously described with some modifications [55 (link),56 ]. In brief, deparaffinized slides were heated in Antigen Retrieval Solution (Cat# ab93678, Abcam, Cambridge, MA, USA) for 15 min, and then, were incubated with 3% hydrogen peroxide in methanol for 10 min. To block any non-specific binding protein, the prepared slides were incubated with Sea-block solution (Cat# 37527, Abcam, Cambridge, MA, USA). Primary antibody was diluted in DAKO diluent solution (Cat# S080983-2, Agilent, Santa Clara, CA, USA) and incubated overnight at 4 °C in a humidified chamber. After washing the slides in PBS, they were incubated with secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The slides were incubated with DAB solution and then counterstained with hematoxylin (Leica, Buffalo Grove, IL, USA). The antibody information is provided in Table S2 .
6
Immunohistochemistry of Lacrimal Gland
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A 5 μm section of the lacrimal gland was deparaffinized and hydrated. The slides were exposed to antigen retrieval solution (Abcam Inc., Cambridge, UK), and peroxidase blocking was performed using 3 % H2O2 solution (Sigma-Aldrich Chemical Co.) for 30 min. The slides were incubated with primary antibodies against F4/80 (catalog No. ab300421, Abcam Inc.), cluster of differentiation 4 (CD4; catalog No. NBP1–19,371, Novus Biologicals, Littleton, CO, USA), interleukin-6 (IL-6; catalog No. ab290735, Abcam Inc.), interleukin-17 (IL-17; catalog No. NBP1–76,337, Novus Biologicals), and tumor necrosis factor-alpha (TNF-α; catalog No. ab1793, Abcam Inc.) for 1 h at room temperature. The slides were then washed and incubated with the secondary antibodies (DAKO Corp., Glostrup, Denmark) for 30 min. The slides were visualized using the chromogen diaminobenzidine and counterstained with Mayer's hematoxylin (Sigma-Aldrich Chemical Co.). Images of the sections were photographed using the EVOS FL Auto 2 imaging system (Thermo Fisher Scientific).
7
Histological Analysis of Lung Tissue
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Histological analysis was performed as described previously (Kwon et al., 2018 ). Lung was fixed in 4% formalin and embedded in paraffin. The sections of 5 μm thickness were cut by microtome (Leica RM2125, Leica Biosystems, Heidelberg, Germany) and were stained with H&E. For immunohistochemistry analysis of the lung tissue, the sections of 5 μm thickness were deparaffinized, rehydrated, cooked in antigen retrieval solution (Abcam, Inc.), and dipped in 3% hydrogen peroxide solution for 30 min. Tumor necrosis factor alpha (TNF‐α; Cat No. ab6671, Abcam, Inc.) antibody was then applied and incubated for 1 hr at RT. After washing, the sections were incubated with secondary antibody (DAKO Corp, Glostrup, Denmark) for 40 min. Immunoreactions were visualized with diaminobenzidine chromogen, and the sections were counterstained with Mayer's hematoxylin (Sigma‐Aldrich Chemical Co.) for 30 s at RT. Images of the sections were photographed with microscope (Carl Zeiss).
8
Histological and Immunohistochemical Analysis of Liver
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The liver samples were fixed in 4% formaldehyde for 2 days and embedded in paraffin. Sections of blocks were cut at 4 μm thickness using a microtome (Leica RM2125; Leica Biosystems, Heidelberg, Germany). For hematoxylin and eosin (H&E) and Sirius red staining, the sections were deparaffinized, rehydrated, and washed with distilled water to allow penetration of the hydrophilic solution. After tissue watering, H&E staining was performed to observe histological changes in the liver sample. For immunohistochemistry analysis, the sections were deparaffinized, rehydrated, cooked in antigen retrieval solution (Abcam), and dipped in 3% hydrogen peroxide solution for 30 minutes. Specific primary antibodies were applied for 1 hour at room temperature, and the sections were incubated with secondary antibodies for 40 minutes. Immunoreactions were visualized with 3,3’-diaminobenzidine staining, and the sections were counterstained with Mayer’s hematoxylin. All data were normalized against the equivalent data in mice fed chow (control). Immunostaining was quantified using ImageJ software (ImageJ software, 1.52a; National Institutes of Health, Bethesda, MD, USA).
9
Immunohistochemical Analysis of Lacrimal Gland
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For immunohistochemical analysis, 5-μm thick sections of the lacrimal gland and eyeball were deparaffinized, hydrated, processed in antigen retrieval solution (Abcam, Inc., Cambridge, UK) and exposed to 3% H2O2 solution (Sigma-Aldrich Chemical Co.) for 30 min. The slides were incubated with primary antibodies against cluster of differentiation 3 (CD3, Abcam, Inc.), CD4 (Novus Biologicals, Littleton, CO, USA), F4/80 (Abcam, Inc.), interleukin-6 (IL-6, Abcam, Inc.), interleukin-17 (IL-17; Abcam, Inc.) and tumor necrosis factor alpha (TNF-α; Abcam, Inc.) for 1 h. Subsequently, the sections were incubated with secondary antibodies (DAKO Corp, Glostrup, Denmark) for 40 min, followed by probing with diaminobenzidine chromogen, and counterstained with Mayer’s hematoxylin (YD Diagnostics Co.). The stained slides were photographed using an imaging system (Thermo Fisher Scientific). The quantitative analysis of histological staining performed using “threshold tool” of ImageJ® (National Institutes of Health, Bethesda, MD, USA).
10
Histological Analysis of Liver Samples
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This analysis included 7 control subjects, 7 AD patients, 7 were fixed in 4% formaldehyde for two days and embedded in paraffin. The section of blocks was cut into thickness of 4 µm musing a microtome (Leica RM2125, Leica Biosystems, Heidelberg, Germany). For hematoxylin and eosin (H&E), Sirius red staining the sections were deparaffinized rehydrated, and washed with distilled water to allow the hydrophilic solution to penetrate. After tissue watering, H&E staining was performed to observe liver sample histological changes. For IHC analysis, the sections were deparaffinized, rehydrated, cooked in antigen retrieval solution (Abcam, Cambridge, UK), and dipped in 3% hydrogen peroxide solution for 30 minutes. The primary antibody used was anti-COL6A6 (1:200, ab150926; Abcam) were then applied for 1 hour at room temperature, and the sections were incubated with secondary antibodies for 40 minutes. Immunoreactions were visualized with DAB staining, and the sections were counterstained with Mayer’s hematoxylin. All data were normalized against the equivalent data in mice fed chow (control). Immunostaining was quantified using ImageJ software (ImageJ software, 1.52a; National Institutes of Health, Bethesda, MD, USA).
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