The sections were deparaffinized in xylene, hydrated in graded ethanol, and washed for 5 min in tap water. The sections were rinsed in distilled water and treated for antigen retrieval using a citrate buffer. Then, immunohistochemical analysis using a Vectastain ABC Kit (Vector Laboratories, USA) was performed according to the manufacturer's instructions, and the sections were incubated with normal blocking serum and the primary antibodies LC3 (#3868; Cell Signaling Technology, USA) and p62 (ab56416; Abcam, UK) overnight. The next day, the sections were incubated for 1 h with a biotinylated secondary antibody. The sections were examined using the ImmPACT NovaRED peroxidase substrate (Sk‐4805; Vector Laboratories, USA).
Immpact novared peroxidase substrate
Manufactured by Vector Laboratories
Sourced in United States
ImmPACT NovaRED Peroxidase Substrate is a chromogenic substrate for the detection of peroxidase enzyme activity in immunohistochemical and other applications. It produces a red-brown reaction product upon oxidation by peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain abc kit, by Vector Laboratories (2 mentions)
Automated slide stainer, by Agilent Technologies (2 mentions)
K4003, by Agilent Technologies (2 mentions)
Anti hsp90 antibody, by Cell Signaling Technology (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Ab66155, by Abcam (1 mentions)
Ab28364, by Abcam (1 mentions)
A0452, by Agilent Technologies (1 mentions)
Cst9661, by Cell Signaling Technology (1 mentions)
Ab92547, by Abcam (1 mentions)
Biotinylated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bovine testicular hyaluronidase, by Merck Group (1 mentions)
Dako antibody diluent, by Agilent Technologies (1 mentions)
Dako envision system hrp labelled polymer detection kit, by Agilent Technologies (1 mentions)
Antigen retrieval solution, by BioGenex (1 mentions)
Mayer s hematoxylin, by Agilent Technologies (1 mentions)
H 5000, by Vector Laboratories (1 mentions)
Ez dewax, by BioGenex (1 mentions)
Horse serum, by Vector Laboratories (1 mentions)
17 protocols using immpact novared peroxidase substrate
1
Immunohistochemical Analysis of Autophagy Markers
2
Immunohistochemical Detection of Hsp90
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Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of alcohol. Antigens were retrieved with 0.01 M citrate buffer (pH 6.0) by heating the samples in a microwave oven for 10 min. The tissue sections were then placed in 3% hydrogen peroxide for 5 min to inactivate endogenous peroxidase, and blocked for 30 min with normal horse serum (VECTASTAIN Elite ABC kit; Vector Laboratories, Burlingame, CA, USA). The primary antibody used for this study was anti-Hsp90 antibody (Cell Signaling). Pre-diluted primary antibodies were applied overnight at 4°C. The slides were then treated with biotinylated secondary antibody for 30 min at room temperature, followed by immPACT NovaRED Peroxidase substrate (Vector Laboratories) for 5 min at room temperature.
3
HLA-A Immunohistochemistry Scoring Protocol
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Immunohistochemistry (IHC) was performed on FFPE tissue as describe before47 (link). In short antigens were retrieved using a citrate buffer (Retrieval solution, cat. no. S1699, pH 6, Dako, Glostrup, Denmark) in a steamer at 100 °C for 30 minutes. Antibodies used in this study are listed in Supplementary Table S1 . Samples were incubated for 1 hour with an HLA-A specific antibody (clone EP1395Y, Abcam, Bristol, UK) diluted 1:1000 in antibody diluent (Dako). After incubation with a biotinylated secondary antibody and addition of streptavidin peroxidase, the detection was obtained using ImmPACT NovaRED Peroxidase Substrate (Vector Laboratories, Burlingame, CA, USA). Three independent investigators (CR, DS, JCB) classified the staining intensity on a scale from zero to three and the percentage of HLA-A positive cells as follows: 0% = 0, 1–25% = 1, 26–50% = 2, 51–75% = 3, >75% = 4. The HLA-A staining score was calculated by multiplying the score for staining intensity with the percentage of positive cells (min = 0, max = 12).
4
Immunohistochemical Analysis of Cellular Markers
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Tissues were fixed in formalin for 24 hours and paraffin-embedded. Four micron sections were deparaffinized and antigen retrieval was performed with either citrate buffer, pH 6 (for Ki67) or Tris-EDTA buffer, pH 9 (for caspase-3, CD3 and CD31). Sections were boiled in retrieval buffer for 20 minutes, then cooled for 20 minutes. Using an automated slide stainer (Dako Australia, North Sydney, NSW, Australia) sections were quenched with hydrogen peroxide, incubated with primary antibody then secondary antibody (K4003, Dako) and revealed with diaminobenzidine (Dako) or ImmPact NovaRed Peroxidase Substrate (Vector Laboratories, Burlingame, CA). Sections were counterstained in Mayer's hematoxylin. Primary antibodies were: Ki67 (ab66155, Abcam, final dilution 1:250), cleaved caspase-3 (CST9661, Cell Signaling Technology, Beverly, MA, final dilution 1:300), CD3 (A0452, Dako, final dilution 1:400) and CD31 (ab28364, Abcam, final dilution 1:100). Images were analyzed using CellProfiler, Broad Institute, MA [59 (link)], quantifying the number of positively stained pixels or total number of cells, identified by hematoxylin staining and the number of diaminobenzidine- or ImmPact NovaRed-positive cells present in the images. See Supplementary Methods for further details.
5
Immunohistochemical Visualization of Mitochondria
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Mouse livers were fixed in 4% paraformaldehyde, paraffin embedded, and sliced into 5 μm sections. Endogenous peroxidases were quenched using 0.3% hydrogen peroxide solution followed by blocking using 2.5% goat serum. Mouse anti‐human mitochondrial IgG (1:1,000; Merck Millipore, Billerica, MA, www.emdmillipore.com ) and horseradish Peroxidase‐Conjugated goat anti‐mouse IgG (1:2,000; Vector Laboratories, Burlingame, CA, vectorlabs.com ) were used as primary and secondary antibodies, respectively. Bound antibodies were visualized using a peroxidase detection kit (ImmPACTNovaRED Peroxidase Substrate; Vector).
6
Immunohistochemical Analysis of Keloid Biopsies
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Keloid biopsies were acquired and immediately fixed in 4% formaldehyde for 30 min. The specimens were then embedded in paraffin. Each slide was cut into 5-μm-thick sections. The slides were stained with different primary antibodies for immunohistochemistry, including mouse anti-E-cadherin (ab1416; 1:1,000), rabbit anti-vimentin (Ab92547; 1:1,000) (both from Abcam, Cambridge, UK), mouse anti-fibronectin (sc-8422; 1:400; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and mouse anti-HIF-1α antibodies (610958; 1:400; BD Biosciences, San Jose, CA, USA). All slides were incubated in biotin-labeled goat anti-rabbit (sc-45101) or goat anti-mouse (sc-395764) serum (1:200; Santa Cruz Biotechnology, Inc.) for 0.5 h, and detection was carried out using the VECTASTAIN Universal ABC-Alkaline Phosphatase kit with ImmPACT NovaRED Peroxidase Substrate (Vector Laboratories, Inc., Burlingame, CA, USA).
7
Immunohistochemical Analysis of Aggrecan in TMJ
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Paraffin-embedded sections of the TMJ at 2, 6, and 9 months of age were stained with rabbit polyclonal antibody against aggrecan neopeptide at 1∶200 dilution (NB-100-74350, Santa Cruz Biotechnology). Sections were reacted with biotinylated secondary antibody (Jackson Laboratories, West Grove, PA) and color was developed with ImmPact NovaRED peroxidase substrate (Vector Labs, Burlingame, CA).
8
BrdU and Filaggrin Immunostaining
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For staining of BrdU-labeled nuclei, deparaffinized and rehydrated 5-μM sections were incubated in 3% hydrogen peroxide for 10 minutes to quench peroxidase activity, blocked in 2% bovine serum albumin in PBS with 0.1% Tween-20 for one hour, and incubated with 1:200 dilution of BrdU (ZBU30) antibody (03–3900, Invitrogen) at 4 ⁰C overnight. Slides were washed in Tris-buffered saline with 0.1% Tween-20 (TBST) and incubated with a 1:50 dilution of MultiLink biotinylated secondary antibodies (SuperSensitive LinkLabel-IHC Detection Kit, Biogenex, San Ramon, CA) for 30 min at room temperature. Next, slides were washed with TBST and incubated with 1:50 dilution of streptavidine-conjugated horseradish peroxidase label followed by washes and incubation with ImmPACT NovaRed Peroxidase Substrate (Vector Laboratories). For staining against filaggrin, an antigen retrieval step was included after rehydration of the sections. Sections were submerged in 10 mM sodium citrate, pH 6.0, preheated to 65 ⁰C, incubated overnight and processed as above. Mouse monoclonal antibody against filaggrin (NCL-FILAGGRIN, Leica Microsytems Inc. Bannockburn, IL) was used at a 1:200 dilution.
9
Quantification of Cartilage MMP-13 Expression
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Immunostaining was performed as previously described.31 (link) Briefly, 4 µm sections (intervening levels between the sections used for pathology scoring) from 5 to 8 randomly selected mice from each group, were dewaxed, rehydrated and digested with 500 U/mL bovine testicular hyaluronidase (Sigma-Aldrich, St. Louis, MO) before overnight incubation with polyclonal rabbit anti-rat matrix metalloproteinase (MMP)-13 that cross reacts with mouse32 (link) (LSBio, LS-B3168, 1.5 µg/mL), in Dako antibody diluent (#S0809, Agilent Technologies) or isotype-matched IgG. For immunodetection, Dako EnVision+System HRP labelled polymer detection kit (Dako) was used with ImmPACT NovaRED Peroxidase Substrate (Vector Laboratories, Burlingame, California, USA), counterstained by Mayer’s haematoxylin and Scott’s bluing solution, and digital images acquired (Nano Zoomer). All samples were stained simultaneously to exclude between-run variability. The number of MMP-13 positive chondrocytes in anterior, central and posterior regions of tibial and femoral non-calcified and calcified cartilage (demarcated by tidemark) were counted (average from two independent scorers), summed and presented as either total tibial, femoral or tibiofemoral. The percentage of a standard ROI in the anterior tibial synovial fossa stained for MMP-13 was quantified using ImageJ.
10
Immunohistochemical Analysis of Liver Tissue
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The methods are described in detail in the Supporting Information. Liver sections were deparaffinized in EZ‐DeWax (HK 585‐5K; BioGenex, San Ramon, CA) and antigen retrieval was performed in antigen retrieval solution (HK 086‐9K; BioGenex). Slides were treated sequentially with peroxidase suppressor, universal block, and avidin (36000; Thermo Fisher Scientific, Waltham, MA). Slides were incubated sequentially with primary antibody diluted in universal block containing a biotin, biotinylated goat antimouse IgG, and avidin‐biotin complex. Slides were developed with Immpact Nova Red peroxidase substrate (SK‐4805; Vector Labs, Burlingame, CA), counterstained with Mayer’s Hematoxylin (S3309; Agilent Dako, Santa Clara, CA), dehydrated, and mounted in nonaqueous mounting media (H‐5000; VectorLabs). Ki67 was detected with a mouse monoclonal antibody (M7240, MIB‐1; Agilent Dako) at 1:100 dilution and activated caspase 3 was detected with a rabbit antibody (9664S, 5A1E; Cell Signaling Technology, Danvers, MA) at 1:100 dilution.
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