Immunohistochemical stainings were performed as described in19 (link). Primary antibodies used are anti-Troponin T cardiac isoform (Thermo Scientific); anti-CD45, α-SMA, anti-Cytokeratin 10 and anti-Ki 67 (all from Abcam). For detection of binding of the primary antibody, the Ultra-Sensitive ABC Peroxidase rabbit/mouse IgG staining kit (Thermo Scientific, Waltham, MA, USA) and the ImmPACT VIP-Peroxidase substrate kit (Vector, Burlingame, CA, USA) were used according to the respective manufacturer’s protocols. Nuclei were counterstained with hematoxylin and slides were dehydrated, cleared in xylene, and mounted with Eukitt (Sigma Aldrich, USA).
Immpact vip peroxidase substrate kit
Manufactured by Vector Laboratories
Sourced in United States, Canada
The Immpact VIP peroxidase substrate kit is a laboratory product designed for use in immunohistochemistry and other enzyme-linked assays that utilize peroxidase as the reporter enzyme. The kit provides a chromogenic substrate that produces a purple-colored reaction product when catalyzed by the peroxidase enzyme, allowing for the visualization and detection of target proteins or other analytes.
Automatically generated - may contain errors
Lab products found in correlation
α sma, by Abcam (1 mentions)
Anti cd45, by Abcam (1 mentions)
Anti cytokeratin 10, by Abcam (1 mentions)
Anti ki67, by Abcam (1 mentions)
Eukitt, by Merck Group (1 mentions)
Apoptag peroxidase detection kit, by Merck Group (1 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Dpx mounting medium, by Merck Group (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
Pannoramic scanner, by 3DHISTECH (1 mentions)
Sigmafast 3 3 diaminobenzidine tablets, by Merck Group (1 mentions)
5 protocols using immpact vip peroxidase substrate kit
1
Immunohistochemical Staining Protocol
2
Quantifying Apoptosis in Liver Tissues
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Apoptotic cells in liver tissues were identified by labeling and detecting DNA strand breaks by the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling method (TUNEL) using the ApopTag Peroxidase Detection kit (Millipore, Temecula, CA) according to the manufacturer’s instructions. Stained apoptotic cells with hepatocyte morphology were counted by an experienced pathologist masked to the treatment groups. Fifteen high‐power fields on each tissue section were used for counting. To detect apoptotic cells with HSC morphology, TUNEL‐stained liver tissues were subsequently stained with the antibody to desmin (a marker of HSC) as described in Materials and Methods, except that secondary antibody peroxidase activity was detected with the ImmPACT VIP Peroxidase Substrate kit (Vector Laboratories). This kit produces a purple product that is easily distinguished from the brown product of the TUNEL staining. Tissues were counterstained with Contrast Green (SeraCare Life Sciences, Milford, MA). Cells costained for TUNEL and desmin were counted on fifteen 200× fields per each tissue slide by an investigator masked to treatment groups.
3
Immunohistochemical Analysis of Lung Proteins
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After euthanizing the mice, the left lung was fixed in 4% paraformaldehyde; immunohistochemistry (IHC) was performed on paraffin-embedded lung tissue sections. In brief, the paraffin-embedded lung sections were deparaffinized with xylene, and the tissue sections were subjected to heat-induced antigen retrieval in a Tris buffer (10 mM Tris–HCl buffer at pH 9). Furthermore, endogenous peroxidase activity was quenched by incubating the sections in 3% hydrogen peroxide for 20 min, and the tissue sections were blocked with 10% goat serum for 20 min. The sections were incubated with specific primary antibodies (BiP, p-JNK, Erp57, and Lc3b) overnight at 4°C. The following day, the sections were probed with goat anti-rabbit antibodies (HRP-conjugated). Finally, detection of protein was carried out by using the Immpact VIP peroxidase substrate kit (Vector Laboratories, Burlingame, CA) (Fukumoto et al., 2019 (link)). The sections were imaged by using a microscope (Olympus BX43, Tokyo, Japan), attached to an Olympus DP21. The images were processed using Adobe Photoshop ver C56.
4
Tissue Preparation and Slide Mounting
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Slides were removed from the 42 °C hybridization oven and dipped in 2× SSPE to aid in coverslip removal. Once coverslips were removed slides were washed in 2× SSPE for 30 min at room temperature, 1× SSPE for 30 min at 52 °C, and blocked for 30 min at room temperature using 1× Blocking Reagent. Samples were treated with Streptavidin-HRP at a concentration of 1:2500 (Invitrogen Carlsbad, CA.) in 1× PBS for 1 h at room temperature, and washed three times in 1× PBS, prior to application of ImmPACT™ VIP Peroxidase Substrate Kit (Vector Laboratories) following manufacturer’s instructions. To rinse and dehydrate tissue samples slides were placed consecutively into DEPC-treated water for 5 min, and 50 % ethanol and 100 % ethanol for 3 min each. Slides were allowed to dry completely before application of Permaslip Mounting Medium and Liquid Coverslip Solution (American MasterTech, Lodi, CA). Immediately following the addition of mounting media, a new coverslip was applied to permanently mount and preserve the tissue sections.
5
Immunohistochemical Analysis of Immune Cells
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