Frozen sections (5 μm) from the brains of adult patients with MS were double-stained for collagen I, IV, or V and for GFAP as previously described [6 (link)]. Cryosections were initially treated with Bloxall (Vector Labs) to remove endogenous peroxidase and alkaline phosphatase, then incubated with primary antibodies against collagen I, IV, or V as described above, and the reaction was developed with Nova RED (Vector Labs) for collagen I and RGC-32 or Vector alkaline phosphatase substrate kit III (Vector Labs) for collagens IV and V. The sections were then incubated overnight with mouse IgG anti-GFAP monoclonal antibody (eBioscience, San Diego, CA). The slides were washed several times in PBS and reacted with alkaline phosphatase-conjugated goat anti-mouse (Sigma-Aldrich), diluted 1/400 and processed with Vector alkaline phosphatase substrate kit III (Vector Labs) or Nova RED as the chromogen substrate.
Novared
Manufactured by Vector Laboratories
Sourced in United States, Canada, United Kingdom
NovaRed is a chromogenic substrate for the detection of peroxidase activity in immunohistochemistry and Western blotting applications. It produces a red-colored reaction product when cleaved by peroxidase enzymes.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Vector Laboratories (5 mentions)
Asp300, by Leica (5 mentions)
Rm2235, by Leica (5 mentions)
Ds fi1, by Nikon (5 mentions)
Citra plus solution, by BioGenex (5 mentions)
Biotinylated secondary antibody, by Vector Laboratories (5 mentions)
Autostainer, by Agilent Technologies (4 mentions)
Vector blue, by Vector Laboratories (4 mentions)
Immpress ap, by Vector Laboratories (4 mentions)
Thimerosal, by Merck Group (3 mentions)
Avidin biotin peroxidase, by Vector Laboratories (3 mentions)
Gill s hematoxylin, by Thermo Fisher Scientific (3 mentions)
Vectastain elite abc kit, by Vector Laboratories (3 mentions)
Impress universal kit, by Vector Laboratories (3 mentions)
Immpact dab, by Vector Laboratories (3 mentions)
Vectastain elite abc reagent, by Vector Laboratories (3 mentions)
Bloxall, by Vector Laboratories (3 mentions)
Ab150678, by Abcam (3 mentions)
Horse serum, by Vector Laboratories (3 mentions)
Duolink in situ pla, by Merck Group (3 mentions)
137 protocols using novared
1
Collagen and GFAP Staining in MS Brains
2
Immunohistochemical Profiling of Tissue Samples
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For immunohistochemistry (IHC) staining, the following antibodies were applied on formalin-fixed, paraffin-embedded tissue using a routine immunoperoxidase technique [15 , 16 ] and experimental sequential IHC [17 (link)]: anti-CD3 (Dako M7254), anti-CD4 (Dako M7319), anti-CD8 (Dako M7103), anti-CD20 (Dako M0755), anti-Bcl2 (DakoM0887), anti-MPO (Dako C7246), anti-CD61 (Dako C7280). Two chromogens were used, DAB (DAB chromogen) as fix and NovaRed (VECTOR NovaRed) as removable chromogen for sequential IHC [17 (link)].
3
Apoptosis Detection in Tissue Sections
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Immunohistochemical staining was performed using TACS 2 Tdt-Fluor In Situ Apoptosis Detection Kit (Trevigen; Burlington, Canada). Briefly, after sections were de-paraffinized, they were incubated with Proteinase K (1:50 in ddH2O) for 30 min at 37 °C. Endogenous peroxidase quenching was performed for 5 min. Slides were coated in Labeling Reaction Mix at 37 °C for 1 h and immersed in 1× TdT Stop Buffer to stop the labeling reaction. Slides were then incubated with horseradish peroxidase-streptavidin (Vector Laboratories; Burlington, Canada), followed by Nova Red (Vector Laboratories) and haematoxylin (Sigma Aldrich). Immunofluorescent TUNEL staining was performed as previously [7 (link)], using a TMR-in situ cell death detection kit (Roche).
4
Immunohistochemical Analysis of Lung Tissue
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Human LGs from three donors were obtained from Advanced Tissue Services (Phoenix, AZ, USA). The LG were removed ≤24 hours after death. Tissues were preserved immediately in RNAlater and shipped at 4°C overnight. All donors were females, and their ages at the time of death were 62, 84, and 90 years. The LGs were embedded in paraffin, and 5-μm sections were prepared. Endogenous peroxidase activity on rehydrated sections was blocked by treating slides with 3% hydrogen peroxide in absolute methanol for 30 minutes. Antigen retrieval was performed for 40 minutes using 0.01 M citrate (pH 6.39) in a humidified heated chamber. Sections were blocked with 5 g/L casein (Sigma Aldrich) in PBS containing 0.5 g/L thimerosal (Sigma-Aldrich; cat# T5125-25G) for 30 minutes, incubated with primary antibodies, and diluted in casein buffer 1:50 overnight at 4°C. Biotinylated goat anti-rabbit IgG antibodies (Vector Labs, Burlingame, CA, USA) were used at a 1:300 dilution. Visualization was achieved using biotin/avidin-peroxidase (Vector Labs) and Nova Red (Vector Labs). Counterstaining was made with Gill's hematoxylin (Fisher Scientific, San Diego, CA, USA; CS400).
5
Immunohistochemical Analysis of Lacrimal Gland
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Lacrimal glands were surgically excised and immersed in 4% paraformaldehyde overnight at 4°C. The tissue blocks were washed, dehydrated, embedded in paraffin, cut to a thickness of 3 mm. The cells were counted that stained positively for CD4 (clone H129.9, 10μg/mL; BD Bioscience, San Diego, CA), CD8α (clone 53e6.7, 3.125μg/mL; BD Bioscience), CD11b (clone M1/70, 6.25 μg/mL; BD Bioscience),CD45 (clone 30-F11, 10 μg/mL; BD Bioscience), CD103 (clone 2E7, 10 μg/mL; Biolegend, San Diego, CA), paraffin sections were stained with the abovementioned primary antibodies and appropriate biotinylated secondary antibodies (all from BD Pharmingen, San Diego, CA) using a staining kit ( Vectastain Elite ABC kit; Vector, Burlingame, CA) and reagents (Nova-Red; Vector). Secondary antibody alone and appropriate anti-mouse isotype (BD Biosciences) controls were also performed. Two sections from each animal were examined and photographed with a microscope (Imager.Z1; Carl Zeiss Meditec, Oberkochen, Germany). Positively stained cells were counted in the stroma of the LG using image-analysis software (NIS Elements Software, version 3.0 BR; Nikon). Results were expressed as the number of positive cells per mm2.
6
RGC-32 Immunohistochemical Staining Protocol
7
PCNA Immunohistochemistry for Panc02 Tumors
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Panc02 tumors were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. For PCNA immunohistochemical staining, the sections were incubated with anti-PCNA antibody (eBioscience, San Diego, CA, USA) at 4°C overnight. Primary antibody staining was visualized using the ImPress Universal kit (Vector Laboratories, Burlingame, CA, USA) with NovaRed (Vector Laboratories) as a substrate. The sections were then counterstained with hematoxylin. To quantify immunohistochemical staining, we counted the number of positive cell per 20× microscope field with 10 random fields per animal. The data were expressed as the percentage changes in the positive cell number over SE mice.
8
Immunohistochemical Staining of DsRed
9
Immunohistochemical Analysis of Mouse Tissues
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Example 16
Mice were anaesthetized, and spleens and livers were collected, frozen in O.C.T. compound and stored at −80° C. until processed. Six to eight micron sections were cut, air dried, fixed for 10 minutes in acetone at −20° C., air dried for at least 4 hours, re-hydrated in TBS for one hour, blocked in 2% N.S. for 1 hour and incubated with primary antibodies overnight at 4° C. with or without 0.1% saponin depending on the antigen. Then, sections were incubated with HRP-labeled secondary antibodies for 1 hour. Slides were developed with ImmPact DAB or NovaRed substrates (Vector Laboratories), air dried, mounted, and analyzed on a Leica microscope. For immunofluorescence stainings, slides were immediately mounted after washing the secondary antibodies. Confocal imaging was done on a Zeiss 510 Meta Confocal microscope.
10
Immunohistochemical and Immunofluorescence Analysis
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Paraffin-embedded tissue sections were cut at 10-μm-thick. Immunohistochemical (IHC) began with microwave antigen retrieval for 6 min (power 8) using Trilogy buffer (Cell Marque; Rocklin, CA) for CD68 and Declere buffer (Cell Marque; Rocklin, CA) for IBA1. Sections were then placed in 3% H2O2 in methanol for 30 min. Following washes in distilled water, sections were blocked in 5% goat serum at room temperature for 1 h. Sections were incubated in primary antibodies IBA1 (rabbit polyclonal, 1:1,000 IHC, Wako); CD68 (clone KP1) (1:50 IHC, Dako) overnight at 4°C. A biotinylated secondary antibody (Vector Laboratories) was amplified using avidin-biotin substrate (ABC solution, Vector Laboratories catalog no. PK-6100), followed by color development in Nova Red (Vector Laboratories). Immunofluorescence (IF) staining was done following microwave antigen retrieval for 6 min (power 8) using Declere buffer (Cell Marque; Rocklin, CA) for primary antibodies to: IBA1 (rabbit polyclonal, 1:250 IF, Wako); and PHF-1 (1:500 IHC and IF, a kind gift from Dr Peter Davies, Bronx, NY), and visualized using appropriate secondary antibody conjugated to an Alexafluor probe (1:200, Lifetechnologies) applied for 1 h. A 0.1% solution of Sudan Black was used to reduce autofluorescence. Slides were coverslipped using Vectashield mounting medium with DAPI (Vector Labs, Burlingame, CA).
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