Standard immunohistochemistry was performed to assess the CD44 protein expression in the diverse formalin fixed human skin tissues. Briefly, 5 µg sections of paraffin embedded blocks were deparaffinized and hydrated prior to epitope retrieval treatment with HIER citrate buffer (Zytomed Systems, Berlin, Germany) for 20 min at 95°C. Endogenous peroxidases were blocked by incubation with 1% (V/V) H2O2 for 20 min at room temperature, before serum (Agilent, Santa Clara, USA) for 1 h at room temperature and incubated with the primary CD44 antibody overnight at 4°C. Negative controls were incubated solely in TBS-T. After washing steps, the HRP-linked secondary antibody was added. The detection was performed with the DAB liquid kit (Dako, Santa Clara, USA) and finally the reaction was quenched by immersion in tap water. After haematoxylin staining and dehydration, the slices were mounted with Eukitt® mounting medium (ORSAtec, Bobingen, Germany). All applied antibodies used are listed in the Table 4 . The photo documentation was performed with a DMi8 microscope (Leica, Wetzlar, Germany). The CD44s expression was scored by two researchers (J.F. and S.J.B.) as follows: 1 – weak, 2 – medium, and 3 – strong.
Hier citrate buffer
Manufactured by Zytomed Systems
Sourced in Germany
HIER Citrate Buffer is a laboratory reagent used for heat-induced epitope retrieval (HIER) in immunohistochemistry (IHC) procedures. It is a citrate-based buffer solution formulated to facilitate the unmasking of antigenic sites in formalin-fixed, paraffin-embedded tissue samples. The buffer helps to restore the immunoreactivity of target proteins, which can be altered during the fixation process, enabling more effective immunodetection.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Merck Group (2 mentions)
H2o2 solution, by Merck Group (2 mentions)
Decloaking chamber, by Biocare Medical (2 mentions)
Axio imager, by Zeiss (2 mentions)
M2 microscope, by Zeiss (2 mentions)
Fluorescent mounting medium, by Agilent Technologies (2 mentions)
Dmi8 microscope, by Leica (1 mentions)
Dab liquid kit, by Agilent Technologies (1 mentions)
Anti rabbit hrp conjugated secondary antibody, by Biocare Medical (1 mentions)
3 3 diaminobenzidine dab, by Biocare Medical (1 mentions)
Vulcan fast red, by Biocare Medical (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Ab3786, by Merck Group (1 mentions)
Ab28364, by Abcam (1 mentions)
Ab8925, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Sc 32790, by Santa Cruz Biotechnology (1 mentions)
Sc 365992, by R&D Systems (1 mentions)
Ab5694, by Abcam (1 mentions)
7 protocols using hier citrate buffer
1
Immunohistochemical Analysis of CD44 Expression
2
Immunohistochemical Detection of TLR4 and Galectin-3
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3‐μm sections from human core samples or mouse lung were deparaffinized, rehydrated, and then treated with 1.8% (v/v) H2O2 solution (Sigma‐Aldrich) to block endogenous peroxidase. Heat‐induced epitope retrieval was performed in HIER citrate buffer (pH 6.0, Zytomed Systems) in a decloaking chamber (Biocare Medical). To inhibit non‐specific binding of antibodies, sections were treated with a blocking antibody (Biocare Medical). Human sections were incubated at 4°C overnight with a rabbit anti‐TLR4 primary antibody (1:50, Cat. No. ab13556, Abcam), followed by 1 h with an anti‐rabbit HRP‐conjugated secondary antibody (Biocare Medical). Signals were amplified by adding chromogen substrate 3,3′‐diaminobenzidine (DAB; Biocare Medical). Mouse sections were incubated at 4°C overnight with a rabbit anti‐galectin‐3 primary antibody (1:100, Cat. No. sc‐20157, Santa Cruz Biotechnology), followed by 1 h with a Rabbit‐on‐Rodent AP‐Polymer (Biocare Medical). Signals were amplified by adding chromogen substrate Vulcan fast red (Biocare Medical). All sections were counterstained with hematoxylin (Sigma‐Aldrich), dehydrated, and mounted.
3
Immunofluorescence Staining of Lung Tissue Sections
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3 μm murine or human lung tissue sections were deparaffinized and rehydrated followed by heat-induced epitope retrieval using HIER Citrate Buffer (pH 6.0, Zytomed Systems). For the cytospins, cells were fixed by incubation with methanol for 10 min followed by 1 min of acetone. Sections or cytospins were blocked with 5% BSA for 30 min, incubated overnight at 4 °C with primary antibodies and 1 h at room temperature with secondary antibodies (anti-goat Alexa Fluor 568, anti-rabbit Alexa Fluor 488, anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 555, Life Technologies) diluted in 1% BSA. Images at different magnifications were captured using Axio Imager with an M2 microscope (Zeiss) and processed with ImageJ 1.x [58 (link)]. Primary antibodies common for human and murine tissue: Galectin-3 (1:50 sc-32790, Santa Cruz), CD31 (1:50 ab28364, Abcam), ACTA-2 (1:600 ab5694, Abcam), Pro-SPC (1:100 AB3786, Chemicon International). Human tissue: DNER (1:100 AF3646, R&D Systems), CC10 (1:100 sc-365992), NOS2 (1:20 sc-8310). Murine tissue: DNER (1:50 AF2254, R&D Systems), NICD1 (1:25 ab8925, Abcam).
4
Immunohistochemical Analysis of Lung Tissue
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For immunohistochemistry, lungs were fixed in paraformaldehyde and embedded into paraffin. After deparaffinizing in xylene and rehydrating in alcohol, the tissue was treated with 1.8% (v/v) H2O2 solution (Sigma-Aldrich, St. Louis, MO) to block endogenous peroxidase. Heat induced epitope retrieval was performed in HIER Citrate Buffer (pH 6.0, Zytomed Systems) in a Decloaking chamber (Biocare Medical, Concord, CA). To inhibit nonspecific binding of antibodies, tissue slides were treated with a rodent blocking antibody (Biocare Medical). After overnight incubation with primary antibodies against MMP12 (Abcam, Cambridge, UK), CD45R/B220 (BD Pharmingen), CD21 (Novus Biologicals, Littleton, CO), CD3 (Sigma Aldrich), p16 (Santa Cruz, CA) or SIRT1 (Millipore, Schwalbach, Germany) tissue slides were incubated with an alkaline phosphatase-labeled secondary antibody (Biocare Medical). Signals were amplified by adding chromogen substrate Vulcan fast red (Biocare Medical). Slides were counterstained with hematoxylin (Sigma-Aldrich) and dehydrated in xylene. Afterwards, coverslips were mounted.
5
Immunofluorescent Staining of Mouse Lung and Human Tissue
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3‐μm sections from mouse left lung or human core samples were stained as described (John‐Schuster et al, 2016 ). Briefly, sections were deparaffinized, rehydrated, and heat‐induced epitope retrieval undertaken using HIER Citrate Buffer (pH 6.0, Zytomed Systems). Sections were blocked using 5% BSA in PBS and then incubated overnight at 4°C with primary antibody, followed by 1 h with secondary antibody and counterstained with DAPI (1:4,000, Sigma‐Aldrich), mounted in fluorescent mounting medium (Dako), and imaged with a fluorescent Olympus BX51 microscope running cellSens software (Version 1.14, Build 14116, Olympus). Primary antibodies: rat IgG2a anti‐mouse CD45r (1:50, clone: RA3‐6B2, BD Biosciences), rabbit IgG1 anti‐mouse CD3 (1:300, Cat. No. C7930, Sigma‐Aldrich), mouse IgG2b anti‐human/mouse CH25H (1:500, Cat. No. ab76478, Abcam). Secondary antibodies: Alexa Fluor 488 conjugated goat anti‐mouse IgG antibody (1:300, Cat. No. A11001, ThermoFisher Scientific), Alexa Fluor 488 conjugated goat anti‐rabbit IgG antibody (1:300, Cat. No. A11008, ThermoFisher Scientific), Alexa Fluor 568 conjugated goat anti‐rat IgG antibody (1:300, Cat. No. A11077, ThermoFisher Scientific).
6
Immunofluorescence Staining of Galectin-3 and PRMT7
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Three-μm sections from paraffin-embedded human core samples or mouse left lung were deparaffinized and rehydrated, followed by heat-induced epitope retrieval using HIER citrate buffer (pH 6.0, Zytomed Systems). Sections were blocked with 5% BSA for 30 min, incubated overnight at 4 °C with primary antibodies (mouse anti-Galectin 3, 1:50, Cat. No. sc-32790, Santa Cruz; rabbit anti-PRMT7, 1:50, Cat. No. sc-98882, Santa Cruz;) and 1 h at room temperature with secondary antibodies (anti-mouse Alexa Fluor 488, 1:500, Cat. No. A28175 and anti-rabbit Alexa Fluor 568, 1:500, Cat. No. A11011, Life Technologies) diluted in 1% BSA. Nuclei were counterstained with DAPI and the slides were mounted in a fluorescent mounting medium (Dako). Images were captured using Axioimager with an M2 microscope (Carl Zeiss) driven by Zen 2.3 (“blue version”) software (Carl Zeiss).
7
Immunohistochemical Profiling of Rat Liver
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For IHC, formalin-fixed rat liver tissue sections were deparaffinized and rehydrated, then heated at 100℃ for 30 min in a HIER Citrate Buffer (0.01 M), (Zytomed, Berlin, Germany) for antigen retrieval. The sliced tissues were later dealt with Hydrogen Peroxide Block (Thermo, Massachusetts, USA) to block the endogenous peroxidase activity. Next, the slides were blocked with Ultra V Block (Thermo, Massachusetts, USA). After rinsing in wash buffer, the sections were incubated with anti-alpha 1 antitrypsin antibody (Abcam, Cat. No. ab9373, Cambridge, UK), rabbit polyclonal to human serum albumin (Abcam, Cat. No. ab2406, Cambridge, UK), and monoclonal mouse anti-human cytokeratin 18 (Dako, Cat. No. M7010, Santa Clara, USA) for 1 hour at room temperature. The slides were then rinsed in wash buffer and incubated for 15 min with secondary antibody horseradish peroxidase-conjuga-ted (HRP Polymer, Thermo, Massachusetts, USA). Immune complexes were detected through the standard substrate detection of HRP. Last, the slides were stained with hematoxylin and dehydrated in graded alcohols and xylene. Digital images were captured with an Aperio Scanscope System AT (Leica, Wetzlar, Germany).
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