Sections (4 μm thick) were deparaffinized and the endogenous peroxidase activity blocked with 0.5% H2O2 in methanol for 10 minutes. Antigen retrieval was performed where necessary, sections blocked with 5% normal serum, and incubated with the primary antibody for 1 hour, followed by biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) diluted 1:500, and streptavidin‐peroxidase (Vector Laboratories) diluted 1:50. Sections were developed in Nova Red peroxidase substrate (Vector Laboratories) and counterstained with hematoxylin.
Novared peroxidase substrate
Manufactured by Vector Laboratories
Sourced in United States
NovaRED peroxidase substrate is a chromogenic substrate used for the visualization of peroxidase enzyme activity. It produces a bright red precipitate upon enzymatic conversion.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin peroxidase, by Vector Laboratories (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Tragacanth gum, by MP Biomedicals (1 mentions)
Bloxall, by Vector Laboratories (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Antigen unmasking solution, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Eclipse ts100 fluorescence microscope, by Nikon (1 mentions)
Asp300, by Leica (1 mentions)
C11440 digital camera, by Nikon (1 mentions)
Rm2235, by Leica (1 mentions)
Formaldehyde, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Ds fi1, by Nikon (1 mentions)
Total erbb4, by Santa Cruz Biotechnology (1 mentions)
Gill s hematoxylin, by Vector Laboratories (1 mentions)
Nuance spectral unmixing camera, by PerkinElmer (1 mentions)
Inform tissue finder software, by PerkinElmer (1 mentions)
Ncl ki67p, by BD (1 mentions)
15 protocols using novared peroxidase substrate
1
Immunohistochemical Staining Protocol
2
Quantifying Virus Infectivity Using Plaque Assay
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A standard plaque assay was used to determine the infectivity of MP-12 [31] (link). For determining the infectivity of scMP-12 and VRP, Vero-G cells in 6-well plates were inoculated with 400 µl of serially diluted samples and incubated for 1 h at 37°C. After removal of the inocula, cells were incubated with MEM containing 0.6% Tragacanth gum (MP Biomedicals), 5% FBS, and 5% tryptose phosphate broth at 37°C. After 3 days incubation, cells were washed with phosphate-buffered saline (PBS) and fixed with PBS containing 4% paraformaldehyde for 20 min at room temperature. After removing paraformaldehyde and overlays, the cells were permeabilized with 0.1% Triton-X100 and incubated with anti-N rabbit polyclonal antibody, which was generated by injecting a purified, bacterially-expressed fusion protein consisting of glutathione-S-transferase and full-length MP-12 N protein into rabbits, followed by incubation with horseradish peroxidase-conjugated, anti-rabbit IgG antibody. The plaques were visualized with Nova RED peroxidase substrate (Vector Laboratories, Burlingame, CA). This modified plaque assay was also used for observing plaque morphologies of MP-12 in Vero-G cells.
3
Immunohistochemical Analysis of Proliferation and Apoptosis
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Immunohistochemical staining for KI67 and caspase 3 (CASP3) was performed in GC monolayers, spheroids and gingiva ex vivo tissues to visualize proliferating and apoptotic cells, respectively.
Sections obtained from samples prepared for histology were deparaffinized. For KI67 and CASP3 staining, sections were steamed with EDTA at pH 9, followed by a wash with TBS. Subsequent blocking was performed with BloxAll (Vector, Burlingame, CA, USA). Afterward, sections were washed with TBS and incubated for 1 h with primary antibodies for KI67 (RM‐9106‐S1, rabbit monoclonal SP6, 1:200; Thermo Fisher Scientific) or CASP3 (9,661, rabbit polyclonal, 1:100; Cell Signaling, Danvers, MA, USA). Afterward, sections were washed with TBS, incubated with secondary antibodies (VWRKDPVR110HRP, antirabbit; Immunologic, Amsterdam, Netherlands) and washed again with TBS. For detection, NovaRED peroxidase substrate (SK‐4805; Vector, Burlingame, CA, USA) was used. The staining process was performed directly in cell culture plates for GC monolayers and automatically by a Lab Vision Autostainer 360 (Thermo Fisher Scientific) for sections of GC monolayers and gingiva ex vivo tissues. Sections without incubation with primary antibodies served as negative controls. Images were taken in 200‐fold magnification under a light microscope.
Sections obtained from samples prepared for histology were deparaffinized. For KI67 and CASP3 staining, sections were steamed with EDTA at pH 9, followed by a wash with TBS. Subsequent blocking was performed with BloxAll (Vector, Burlingame, CA, USA). Afterward, sections were washed with TBS and incubated for 1 h with primary antibodies for KI67 (RM‐9106‐S1, rabbit monoclonal SP6, 1:200; Thermo Fisher Scientific) or CASP3 (9,661, rabbit polyclonal, 1:100; Cell Signaling, Danvers, MA, USA). Afterward, sections were washed with TBS, incubated with secondary antibodies (VWRKDPVR110HRP, antirabbit; Immunologic, Amsterdam, Netherlands) and washed again with TBS. For detection, NovaRED peroxidase substrate (SK‐4805; Vector, Burlingame, CA, USA) was used. The staining process was performed directly in cell culture plates for GC monolayers and automatically by a Lab Vision Autostainer 360 (Thermo Fisher Scientific) for sections of GC monolayers and gingiva ex vivo tissues. Sections without incubation with primary antibodies served as negative controls. Images were taken in 200‐fold magnification under a light microscope.
4
IHC Staining for Ki67 and p53
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Portions of each tumor were fixed in formalin and embedded in paraffin. Tissue sections were deparaffinized with heat and rehydrated in decreasing alcohol concentrations. After rehydration, antigen retrieval was performed by microwaving slides in Antigen Unmasking Solution (Vector, H-3300). After cooling, endogenous peroxides were inactivated by adding 3% hydrogen peroxide for 10 minutes. Slides were blocked in goat serum in PBS for one hour at room temperature. Ki67 primary antibody (1:500, Abcam, ab66155) and p53 antibody (1:500, Leica Biosystems, CM5) were diluted in goat serum in PBS and incubated on slides overnight at 4°C. Biotinylated Rabbit secondary antibody was diluted in goat serum in PBS and added to the slides for one hour at room temperature. Goat serum, secondary antibody, and developing reagents were from Vector ABC kits (PK-4001). Pigment was developed in the tissues using a NovaRed Peroxidase Substrate (Vector, SK-4800). After development, slides were co-stained in hematoxylin, dehydrated, and mounted.
5
Characterization of Phosphorylated FGF23 Antibody
6
Brain Tissue Histology for Flavivirus Detection
7
Immunohistochemical Analysis of SOX2 Expression
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The IHC assays were conducted to detect the expression profile of SOX2 in resected tumors from the sh-NC group and sh-TUG1 group, following the protocols from a previous report (23 (link)). In brief, resected tumors from the xenograft models were fixed in 3% formaldehyde overnight at 4˚C and embedded in paraffin and then cut into 5-µm sections. The slices were then incubated with 0.3% hydrogen peroxide (H2O2) solution in methanol for 20 min at room temperature to block the activity of endogenous peroxidases. Sections were blocked in 10% horse serum at room temperature for 1 h and then incubated with goat anti-SOX2 antibodies (1:100; cat. no. GT15098; Neuromics) at 4˚C overnight, followed by incubation with peroxidase-conjugated horse anti-goat IgG (cat. no. PI-9500-1; 1:2,000; Vector Laboratories, Inc.) at room temperature for 30 min. Immunoreactivity was measured using NovaRed peroxidase substrate (Vector Laboratories, Inc.) under an Eclipse TS100 fluorescence microscope (Nikon Corporation) at x20 magnification.
8
Immunohistochemistry and Immunofluorescence Protocol
9
Quantifying Brain Tissue Markers
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Mouse brain tissue was fixed in 4% paraformaldehyde in PBS overnight and embedded in paraffin. Tissue sections (5 μm) underwent microwave antigen retrieval in a citrate buffer before immunostaining with the following antibodies: Ki-67 (Leica #NCL-Ki67p, 1:5000), cleaved caspase-3 (CC3) (BD #559565, 1:500), p-EGFR Y1173 (Cell Signaling #4407, 1:250), phosphorylated ErbB4 (pErbB4) Y984 (Cell Signaling #3790, 1:50), or total ErbB4 (Santa Cruz #SC-283, 1:200). Sections were developed using biotinylated secondary antibodies, followed by detection with an Elite ABC kit and NovaRED peroxidase substrate, then counterstained with Gill’s hematoxylin (all from Vector Laboratories). Ki67- and CC3-positive cells were quantified using a Nuance spectral unmixing camera and InForm Tissue Finder software (Perkin Elmer). Quantification of phosphorylated and total ErbB4 was performed using particle analysis in ImageJ [32] (link).
10
Immunohistochemical Detection of GRP78
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Paraffin sections were subjected to deparaffinization and rehydration by immersing them in xylene for three 5 min changes, followed by two 5 min changes of absolute ethanol, followed by 70% ethanol for 5 min. Antigen retrieval was performed using pre-heated citrate buffer (pH-6) heated for 15 min, and the slides were left to cool down for 10 min followed by PBS washing. Then, the slides were kept in 3% H2O2 for 10 min to block endogenous peroxides and rinsed thoroughly with PBS. Undiluted fetal calf serum (FCS) was added to each slide and incubated for 1 hour to block non-specific bindings with the antigen. Anti-GRP78 antibody (Abcam, ab108615) diluted 1 : 50 with FCS was added to each sample and incubated for 1 hour. Slides were rinsed gently with PBS and then EnVisionTM dual link system-HRP (Horseradish Peroxidase) (Dako, North America Inc., CA, USA) was applied and incubated for 30 min. To visualize the antigen-antibody reaction, Nova RED peroxidase substrate (Vector Laboratories Inc., CA, USA) was added to each sample and incubated for 3 min. Then, the samples were washed with distilled water to stop the reaction and counter-stained with Hematoxylin.
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