For Masson’s Trichrome’s staining, kidney tissues were fixed for 48 h at RT in 10% neutral-buffered formalin (NBF), dehydrated, embedded in paraffin blocks, and sectioned at 4 μm. Sections were then stained with Masson’s Trichrome according to the standard protocol and examined by light microscopy. For immunohistochemistry, kidneys were processed as mentioned above. Following dewaxing and antigen retrieval processes (98 °C for 20 min in citrate buffer), kidney sections were permeabilized with 0.1% Triton X-100 (Sigma), incubated with 3% H2O2 (Sigma), and blocked with 2.5% normal horse serum (S-2012, Vector Labs). Kidney sections were incubated with primary antibody (X203 and X209 at 1:250 dilution in PBST) overnight (4 °C) and visualized using an ImmPRESS HRP horse anti-mouse IgG polymer detection kit (MP-7402, Vector Labs) with ImmPACT DAB Peroxidase Substrate (SK-4105, Vector Labs). Hematoxylin (H-3401, Vector Labs) was used to stain the nuclei prior to imaging by light microscopy.
Immpact dab peroxidase substrate
Manufactured by Vector Laboratories
Sourced in United States, Germany
ImmPACT DAB peroxidase substrate is a chromogenic substrate used for the detection of peroxidase activity in immunohistochemistry and other applications. It produces a brown reaction product upon enzymatic conversion.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain elite abc kit, by Vector Laboratories (10 mentions)
Vectastain abc kit, by Vector Laboratories (8 mentions)
Avidin biotin blocking kit, by Vector Laboratories (5 mentions)
Immpress reagent kit, by Vector Laboratories (5 mentions)
Hematoxylin, by Vector Laboratories (4 mentions)
Bloxall, by Vector Laboratories (3 mentions)
Signalstain antibody diluent, by Cell Signaling Technology (3 mentions)
Cytoseal 60, by Thermo Fisher Scientific (3 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (3 mentions)
Hyaluronidase, by Merck Group (3 mentions)
Hematoxylin qs, by Vector Laboratories (3 mentions)
Permount, by Thermo Fisher Scientific (3 mentions)
Harris hematoxylin, by Merck Group (3 mentions)
Histofine simple stain max po, by Nichirei Biosciences (2 mentions)
Imagescope, by Leica (2 mentions)
Bx53 microscope, by Olympus (2 mentions)
Vectastain avidin biotin complex method kit, by Vector Laboratories (2 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (2 mentions)
Hematoxylin, by Agilent Technologies (2 mentions)
Methyl green nuclear counterstain, by Vector Laboratories (2 mentions)
104 protocols using immpact dab peroxidase substrate
1
Masson's Trichrome Staining and Immunohistochemistry of Kidney Tissue
2
CYP11B2 Expression in Injured Muscle
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To evaluate levels of CYP11B2 in acutely injured muscle, immunohistochemistry was performed on muscle from mice sacrificed at 1 (n = 3 Cre− [1M, 2F]), 4 (n = 3 Cre− [3F]), and 7 (n = 3 Cre− [3 M]) days after injury. The sections were incubated with: 1:250 CYP11B2 (rabbit anti-mouse AS 2084) and then incubated with 1:200 HRP-conjugated goat anti-rabbit IgG (Jackson Immuno Research Laboratories, West Grove, PA, 111-035-144) and developed with ImmPact DAB peroxidase substrate (Vector, Burlingame, CA, SK-4105). Since 4 days appeared to have the highest levels of CYP11B2, additional 4 days post-injury sections were analyzed (n = 6 Cre− [2M, 4F], and 6 MRcko [3M, 3F]) (n = 6 untreated [3M, 3F], and 6 spironolactone [3M, 3F]). Images of the CYP11B2 staining were taken on a Nikon (Melville, NY) Eclipse 800 microscope with the 20× objective and ND32 and NCB11 filters with the SPOT digital camera software and composited using Adobe Photoshop (Adobe, San Jose, CA, CS6). The percent area of CYP11B2 infiltration was quantified with the Photoshop (Adobe, San Jose, CA, CS6) paint bucket tool by an individual blinded to the genotypes and treatment as previously described (Hauck et al., 2019 (link)).
3
Galectin-9 Immunohistochemistry Protocol
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Paraffin blocks were sectioned (5 μm) and placed on poly-L-lysine-coated glass slides. The sections were deparaffinized for 1 h at 60°C. To block the activity of endogenous peroxidase, the sections were incubated in methanol containing 0.3% H2O2 for 30 min. Thereafter, every step was followed by three washes in PBS buffer. To block nonspecific antibody binding, slides were incubated for 1 h in 1% BSA in PBS. Sections were incubated with galectin-9 antibody (ab69630, Abcam, Cambridge, MA USA) diluted at 1:400 overnight at 4°C. Then, the sections were incubated with HRP-conjugated anti-rabbit IgG (ab6721, Abcam) for 1 h at room temperature. Staining was visualised by incubating the slides with DAB substrate (ImmPACT DAB Peroxidase substrate, Vector SK-4105, Burlingame, CA USA) according to the manufacturer’s instructions. To distinguish the nuclei, sections were counterstained in haematoxylin solution (HX87960674 Merck, Darmstadt Germany) for 10 sec. Images were obtained using a VENTANA DP200 scanner (Roche Diagnostics, Tucson, AZ USA). The intensity of galectin-9 expression was determined using Image-Pro Image Analysis Software (Media Cybernetics, Rockville, MD USA). The mean density was calculated for each sample and the expression of galectin-9 was reported in lumens.
4
Comprehensive Immunohistochemical Analysis of Murine Tissue
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Tissues were fixed in 4% formalin, embedded in paraffin and sections were cut and mounted onto slides. Staining of mice tissue was performed with the following antibodies: anti-Muc5ac (Abcam #ab3649 1:100), anti-Cytokeratin19 (Abcam #ab52625 1:200), anti-ki67 (Abcam #15580 1:100), anti-Gkn1 (Thermofisher #PA547913 1:100), anti-Tgfbeta (Abcam #ab92486 1:100), anti-CD3 (Bio-Rad #MCA1477 1:250), anti-Ly6g (BD Pharmingen 551459 1:200), anti-CD3 (Abcam #ab21703 100ul), anti-Onecut2 (Abcam #ab28466 1:50), anti-F4/80 (Biorad #MCA497RT 1:200), anti-Tff1 (Abcam #ab190942 1:200), anti-Dcamkl1 (Abcam #ab37994 1:50), and anti-insulin (Dako, A0564, GP, 1:10) overnight at 4 °C.
ImmPACT DAB peroxidase substrate (Vector Laboratories #) and ImmPACT Vector red alkaline phosphatase (Vector Laboratories #VE-SK-5150) were used as substrates. Hematoxylin (Vector Laboratories #H-3404) was used as a counterstain.
We used N-Histofine kit, mousestain kit 414321F when staining with anti-Muc5ac (mouse antibody).
ImmPACT DAB peroxidase substrate (Vector Laboratories #) and ImmPACT Vector red alkaline phosphatase (Vector Laboratories #VE-SK-5150) were used as substrates. Hematoxylin (Vector Laboratories #H-3404) was used as a counterstain.
We used N-Histofine kit, mousestain kit 414321F when staining with anti-Muc5ac (mouse antibody).
5
Histological Analysis of TGFBI and LC3
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Formalin fixed, paraffin-embedded sections were processed using conventional histological techniques, including hematoxylin and eosion (HE) stating, Masson trichrome staining, Congo red staining, thioflavin T staining and von Kossa staining. Congo red birefrinence assay was performed. Paraffin-embedded sections were used for immunohistochemistry. Consecutive 6-um-thick paraffin-embedded sections were deparaffinized, rehydrated, and washed with phosphate-buffered saline. The sections were blocked with 10% goat serum for 30 minutes and then incubated overnight at 4°C with a rabbit polyclonal antibody raised against 208 aa length of recombinant peptides corresponding to Asn199-Gly406 of human TGFBI (Proteintech Group, Inc., Chicago, IL) and an anti-LC3 antibody (MBL, Nagoya, Japan). Immunoreactivity of the anti-TGFBI antibody was visualized with VECTASTAIN Elite ABC KIT and ImmPACT DAB Peroxidase Substrate (Vector Laboratories Inc., Burlingame, CA 94010). Immunoreactivity of the anti-LC3 antibody was visualized with secondary antibody conjugated with Cy3 (Chemicon International, Temecula, CA). After they were washed with PBS, the sections were mounted (Permafluor, Beckman Couter Inc., Miami, FL). Images were observed by a microscope (Axio Imager, Carl Zeiss Inc., Oberkochen, Germany) equipped with a digital camera (Axioncam, Carl Zeiss).
6
Exosome Lysis and Western Blot Analysis
7
ABCG2 Expression Quantification in Tissue Arrays
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Two ninety-dot tissue arrays were purchased from Shanghai Outdo Biotech Company (Shanghai, China). Formalin-fixed, paraffin-embedded sections were deparaffinized by xylene, rehydrated in ethanol and boiled in 10 mM citrate buffer (pH 6.0) for 30 min for antigen retrieval. Endogenous peroxidase was blocked by 3% H2O2 for 10 min for immunoperoxidase labeling. Sections were then incubated at 4°C overnight with primary antibodies against rabbit anti-ABCG2 (1:100, Abcam, Cambridge, UK). Incubation with corresponding secondary antibody (ImmPRESS universal peroxidase reagent, Vector Lab, CA, USA) and the peroxidase-antoperoxidase complex was visualized by DAB kit (ImmPACT DAB Peroxidase Substrate, Vector Lab), as previously described [68 (link)]. Two individuals (B.Y. and J.X.), who had no prior knowledge of the clinical pathologic data of the patients, examined the stained sections independently. Positive membrane ABCG2 staining was assessed by the intensity of stained cells and determined in two categories (negative and positive). The staining intensity was classified from 0 to 3+ as follows: 0, no staining; 1+, < 25% of the section was stained; 2+, 26% - 50% of the section was stained; 3+, > 50% of the section was stained. Scores of 1+, 2+ and 3+ were considered to be positive.
8
Immunohistochemical Analysis of Lung Tissues
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Lung tissues collected from euthanized animals were fixed with 4% paraformaldehyde
phosphate buffer solution (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) and embedded
in paraffin wax. Sections (2–4 µm thick) were cut and stained with haematoxylin and eosin
(HE). Immunohistochemical staining was performed by polymer method using Histofine Simple
Stain MAX-PO (Rat) (NICHIREI BIOSCIENCE INC, Tokyo, Japan) or Histofine Simple Stain
MAX-PO (M) (NICHIREI BIOSCIENCE INC). Endogenous peroxidase activity was blocked by 3%
hydrogen peroxide in methanol. Subsequently, sections were incubated with protein block
serum-free reagent (DAKO, Carpinteria, CA, USA) for blocking non-specific reactions. Then
they were incubated with the antisera for 16 hr at 4°C, as primary immunoreaction. The
antisera from rat (1:1,000) were used for sections of guinea pig and mouse, and the
antisera from mouse (1:1,000) were used for sections of rat. Then they were treated with
Histofine Simple Stain MAX-PO (Rat) for tissues of guinea pig and mouse, and with
Histofine Simple Stain MAX-PO (M) for rat tissues for 30 min at room temperature. For
immunohistochemical detection, 3,3′-diaminobenzidine tetrahydrochloride solution (ImmPACT
DAB Peroxidase Substrate; Vector Laboratories, Burlingame, CA, USA) was used.
phosphate buffer solution (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) and embedded
in paraffin wax. Sections (2–4 µm thick) were cut and stained with haematoxylin and eosin
(HE). Immunohistochemical staining was performed by polymer method using Histofine Simple
Stain MAX-PO (Rat) (NICHIREI BIOSCIENCE INC, Tokyo, Japan) or Histofine Simple Stain
MAX-PO (M) (NICHIREI BIOSCIENCE INC). Endogenous peroxidase activity was blocked by 3%
hydrogen peroxide in methanol. Subsequently, sections were incubated with protein block
serum-free reagent (DAKO, Carpinteria, CA, USA) for blocking non-specific reactions. Then
they were incubated with the antisera for 16 hr at 4°C, as primary immunoreaction. The
antisera from rat (1:1,000) were used for sections of guinea pig and mouse, and the
antisera from mouse (1:1,000) were used for sections of rat. Then they were treated with
Histofine Simple Stain MAX-PO (Rat) for tissues of guinea pig and mouse, and with
Histofine Simple Stain MAX-PO (M) for rat tissues for 30 min at room temperature. For
immunohistochemical detection, 3,3′-diaminobenzidine tetrahydrochloride solution (ImmPACT
DAB Peroxidase Substrate; Vector Laboratories, Burlingame, CA, USA) was used.
9
Immunohistochemical Detection of 4-HNE Adducts
10
Immunohistochemical Analysis of IRF6 Expression
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Tissue microarrays (#MC245c and MC246b, amsbio) were baked for 1 h at 60°C before deparaffinization and rehydration through xylene, ethanol, and deionized (dd) H2O. Tris/EDTA buffer, pH 9.0 at 95°C for 30 min was used for antigen retrieval. Specific binding of primary rabbit polyclonal anti-IRF6 antibody (NBP2-49383, Novus Biologicals, Centennial, CO, USA) was visualized with the ImmPACT DAB Peroxidase Substrate (Vector Laboratories, Newark, CA, USA). Nuclei were counterstained with hematoxylin and slides mounted with Aquatex (Sigma-Aldrich, St. Louis, MO, USA).
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