Sections were dewaxed and rehydrated as previously described [9 (link)], followed by heat-mediated antigenic retrieval using a pH 6.0 citrate buffer (BRD2) or a pH 8.0 EDTA buffer (BRD4). Endogenous peroxidase activity was quenched by incubation of the slides in 3% hydrogen peroxide for 30 min; cell membranes were permeabilised by 0.1% saponin in PBS, and non-specific labelling blocked by 5% normal horse serum (BRD2) or 5% normal goat serum (BRD4) in PBS for 20 min, at room temperature (as for all the following steps). After washing in PBS, sections were incubated for 1 h with mouse anti-human BRD2 (sc-393720; Santa Cruz Biotechnology) at a 1∶2900 dilution (0.069 µg/ml) or with rabbit anti-human BRD4 (ab128874; Abcam) at a 1∶300 dilution (1.577 µg/ml). For negative control slides, normal mouse or normal rabbit non-specific immunoglobulins (Santa Cruz Biotechnology) were used at the same protein concentration as the primary antibodies. After repeated PBS washing, sections were incubated with anti-mouse or anti-rabbit biotinylated antibody (Vector ABC Kit, Vector Laboratories) for 30 min. Sections were then incubated with ABC reagent (Vector ABC Kit, Vector Laboratories) for 30 min and stained with chromogen-fast diaminobenzidine (DAB) for 1–5 min. Finally, the slides were counterstained in haematoxylin and mounted on permanent mounting medium.
Vector abc kit
Manufactured by Vector Laboratories
Sourced in United States
The Vector ABC kit is a laboratory product that provides a set of tools and reagents for performing basic analytical procedures. The kit includes a collection of essential items required to carry out common laboratory tasks, but the specific details and intended use of the kit are not available.
Automatically generated - may contain errors
Lab products found in correlation
Dab chromogen, by Thermo Fisher Scientific (4 mentions)
Biotinylated anti mouse igg secondary antibody, by Vector Laboratories (4 mentions)
Mouse anti neun, by Merck Group (4 mentions)
Vectastain abc kit, by Vector Laboratories (4 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Biotinylated secondary antibody, by Vector Laboratories (3 mentions)
P tau s199, by Abcam (3 mentions)
Vector dab kit, by Vector Laboratories (3 mentions)
Ba 1000, by Vector Laboratories (3 mentions)
Anti par antibody, by Enzo Life Sciences (3 mentions)
Aquatex, by Merck Group (3 mentions)
Anti p histone h3, by Cell Signaling Technology (3 mentions)
Histoclear, by National Diagnostics (2 mentions)
Rabbit monoclonal antibody to cd31, by BD (2 mentions)
Peroxidase substrate solution, by Vector Laboratories (2 mentions)
Alexa fluor 594 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Dab substrate kit, by Vector Laboratories (2 mentions)
Dab reagent, by Vector Laboratories (2 mentions)
Permount solution, by Thermo Fisher Scientific (2 mentions)
56 protocols using vector abc kit
1
Immunohistochemical Detection of BRD2 and BRD4
2
Immunohistochemical Analysis of Alzheimer's Pathology
3
Immunostaining of CD31 in Diabetic Mouse Limb
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Immunostaining was performed as previously described [21 (link)]. Briefly, cryosections of limb in diabetic mice after 3 weeks of ischaemia were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 3% bovine serum albumin in PBS. The sections were incubated with rabbit monoclonal antibody to CD31 (Pharmingen, San Jose, CA, USA) at a concentration of 1:100 for 60 min., washed with PBS, and then incubated with goat anti-rabbit IgG secondary antibody at a concentration of 1:200. The Vector ABC kit (Vector Laboratories, Burlingame, CA, USA) was used according to the manufacturers' instruction and stained with DAB. Nuclei were counter-stained with haematoxylin. All images were captured with a light microscope. Capillary density was calculated in five randomly selected fields and counted by an investigator blind to the study protocol. Capillary density was expressed as number/muscle fibre.
4
Slc22a4 and Slc22a5 Immunoreactivity in SOD1 Mice
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In order to determine changes of Slc22a4 and Slc22a5 immunoreactivity, WT littermate and SOD1 (G93A) mice at 120–150 days of age were perfused via using 4% buffered paraformaldehyde in PBS. Spinal cords were extracted and serially sectioned at 30 μm of thickness by cryostat. Lumbar spinal cord sections (2 sections per animal) were used for immunostaining to detect the level of Slc22a4 and Slc22a5. First, the tissue sections were incubated for 1.5 h in a blocking solution (0.3% Triton-X, 2% goat serum, and 2% donkey serum in 0.1 M PBS) and then further incubated with primary antibodies [Slc22a4 (1:200 dilution) (Cat. No.: H00006583-A01, NOVUS) and Slc22a5 (1:200 dilution) (Cat. No.: ab180757, Abcam, Cambridge, MA, USA,)] in a blocking solution at 4 °C for 24 h. After washing three times with PBS, the tissue slides were processed with Vector ABC Kit (Vector Laboratories, Inc., Burlingame, CA, USA). The Slc22a4 and Slc22a5 signals were developed with DAB chromogen (Thermo Fisher Scientific, Meridian, Rockford, IL, USA). Images were then analyzed via using an Olympus microscope system (Olympus, Tokyo, Japan). The semi-quantification of immunoreactivity was analyzed by NIH ImageJ. Especially, the intensity of Slc22a4 and Slc22a5 signals was counted from 5~10 motor neurons per section.
5
Semiquantitative AMPK Immunohistochemistry
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For AMP-activated protein
kinase semiquantitative assessment, the
slides were incubated overnight at 4 °C with primary AMPK antibodies
in the concentrated form consisting of rabbit IgG in phosphate buffer
saline (free from Mg2+ and Ca2+), pH 7.4, 150
mM NaCl, 0.02% sodium azide, and 50% glycerol. Detection was carried
out using a Vector ABC kit (Vector Laboratories, CA) and DAB reagent
(Dako Comp, Japan). All slides were examined as described inSection 2.3.4.3 .
The proportions of positive cells were also calculated, and ranges
were assigned from 10 to 100%. The intensity of staining was given
scores of 0 (negative), 1 (very weak), 2 (weak), 3 (moderate), and
4 (intense). Lastly, the immunoreactivity score was calculated as
a percentage of positive cells multiplied by the intensity of staining.58 (link)
kinase semiquantitative assessment, the
slides were incubated overnight at 4 °C with primary AMPK antibodies
in the concentrated form consisting of rabbit IgG in phosphate buffer
saline (free from Mg2+ and Ca2+), pH 7.4, 150
mM NaCl, 0.02% sodium azide, and 50% glycerol. Detection was carried
out using a Vector ABC kit (Vector Laboratories, CA) and DAB reagent
(Dako Comp, Japan). All slides were examined as described in
The proportions of positive cells were also calculated, and ranges
were assigned from 10 to 100%. The intensity of staining was given
scores of 0 (negative), 1 (very weak), 2 (weak), 3 (moderate), and
4 (intense). Lastly, the immunoreactivity score was calculated as
a percentage of positive cells multiplied by the intensity of staining.58 (link)
6
Immunohistochemical Analysis of nNOS in Mouse BLA
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Immunohistochemistry was conducted as we have previously described (Wang et al., 2017 (link)). Adult mice were intracardially perfused with 4% paraformaldehyde and the brain was removed immediately. The BLA was postfixed for overnight, successively placed in 30% sucrose in 0.1 M phosphate-buffered saline (PBS) overnight and cut on a sliding microtome (40 μm). Free floating sections were quenched for 30 min in 3% H2O2/30% methanol in PBS and incubated in a blocking solution (5% normal goat serum and 2.5% bovine serum albumin in PBST (0.25% Triton X-100)) for 30 min at room temperature. Sections were then incubated with the goat anti-rabbit nNOS (1:5000; Sigma) in the blocking serum at 4°C overnight. After rinsing in PBST (three times for 10 min), sections were reacted for 2 h at room temperature with biotinylated secondary antibody goat anti-rabbit IgG (1:500, Vector Laboratories; Burlingame, CA, USA) and incubated with avidin biotin peroxidase complex (Vector ABC Kit, Vector Laboratories). The reaction was visualized by detection solution (0.25 mg/ml 3,3′-diaminobenzidine in combination with 0.03% H2O2 in PBS). Sections were then transferred onto mounted on glass slides, dehydrated with ascending alcohols, rinsed in xylene and cover slipped.
7
Immunohistochemical Analysis of Tau Phosphorylation
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Immunohistochemistry was performed as previously described.23 (link) Paraffin-embedded tissues were sectioned in a coronal plane at 10–20 μm. The tissue sections were rehydrated, blocked with blocking solution (1% H2O2), and incubated with p-Tau (S202/T205) (1:200), PP2B/PPP3CA (1:200; SantaCruz Biotech), p-Tau (S199) (1:200; Abcam) and anti-III tubulin antibody (1:500 dilutions; Sigma) for 24 h. After washing three times, the slides were processed with Vector ABC Kit (Vector Lab). The immunoreactive signals were developed with DAB chromogen (Thermo Fisher Scientific, Meridian, Rockford, IL, USA) and analyzed under a bright field microscope.
8
Immunohistochemical Analysis of Placental Tissue
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Following fixation in 4% PFA, placenta tissue was processed, and paraffin embedded following standard protocols. 5 µm thick tissue sections were obtained using a microtome (Leica), de-waxed and rehydrated as standard. Targeted antigen retrieval was performed by boiling sections in 1x IHC antigen retrieval solution (Invitrogen) for 15–20 min, followed by a 20 min cooling period at room temperature. Incubation in 3% hydrogen peroxidase solution for 10 min was used to suppress endogenous peroxidase activity, and a protein block of 10% goat serum with 1% bovine serum albumin (BSA; Jackson ImmunoResearch) was then applied for 1 h at room temperature. Primary antibodies, Claudin5, P-glycoprotein, Occludin, Vimentin, and Cytokeratin-7, were diluted in 10% goat serum with 1% BSA and applied to sections overnight a 4°C (antibody manufacturer and concentration listed in Supplementary Table S1 ). Sections were then incubated with a biotinylated secondary antibody for 1 h at room temperature, amplified using the Vector ABC kit (Vector), and detected using DAB (Vector). Hematoxylin was used to counterstain nuclei. Details on antibodies and dilutions are supplied in Supplementary Table S1 . Stained sections were visualized using the Axioscan scanning microscope (Zeiss), and representative images obtained using the Zen imaging software (Zeiss).
9
Immunostaining of Murine Limb Ischemia
10
Immunohistochemical Labeling of Neuronal Nuclei
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Cryostat sections (40-μm thickness) were pre-incubated with blocking buffer (2% goat serum, 0.1% bovine serum albumin and 0.3% Triton X-100 in 0.1 M PB) at room temperature, and then incubated with mouse anti-NeuN (1 mg/ml; Millipore, Temecula, CA) overnight. After washing, the sections were incubated with biotinylated anti-mouse IgG secondary antibody (5 mg/ml; Vector laboratories, Burlingame, CA) for 2 hrs. Sections were visualized by the DAB method, using ABC reagents using a Vector ABC kit (Vector laboratories) and peroxidase substrate solution (Vector laboratories). For fluorescence images, sections were incubated with Alexa Fluor 594-conjugated donkey anti-mouse IgG (Life Technologies Corporation, Grand Island, NY; 1:1000).
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