Immunohistochemistry was performed to detect and count the number of cells in conjunctival epithelium that stained positively for CD4 (clone H129.9, 10 μg/mL, BD Bioscience, San Diego, CA) and appropriate biotinylated secondary antibody (BD Pharmingen) and Vectastain Elite ABC using NovaRed reagents (Vector, Burlingame, CA) as previously described (3 (link)). Secondary antibody alone and appropriate anti-rat isotype (BD Biosciences) controls were also examined. Positively stained cells were counted in the goblet cell rich area of the conjunctiva using image-analysis software (NIS Elements Software, Nikon, Melville, NY) using a ×10 objective. Three sections from three animals were examined (N = 9) for each time point/mouse for CD4+ T cell. Immunohistochemistry was performed to detect the presence of NKG2D positive cells in the conjunctival epithelium with anti-NKG2D antibody (clone 191004, MAB1547, 10μg/mL, R&D Systems) and appropriate biotinylated secondary antibody (BD Pharmingen) and Vectastain Elite ABC using NovaRed reagents (Vector, Burlingame, CA) as previously described above.
Vectastain elite abc
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom, Canada
The Vectastain Elite ABC is a high-sensitivity avidin-biotin complex (ABC) detection system. It is designed to enhance the visualization of target antigens in immunohistochemical and in situ hybridization procedures.
Automatically generated - may contain errors
Lab products found in correlation
Avidin biotin blocking kit, by Vector Laboratories (4 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (4 mentions)
3 3 diaminobenzidine (dab), by Merck Group (4 mentions)
Dab peroxidase hrp substrate kit, by Vector Laboratories (3 mentions)
Dab substrate kit, by Vector Laboratories (3 mentions)
Novared reagents, by Vector Laboratories (3 mentions)
Ab5457, by Merck Group (3 mentions)
Mab5280, by Merck Group (3 mentions)
Ba 9200, by Vector Laboratories (3 mentions)
Biotinylated secondary antibody, by Vector Laboratories (3 mentions)
Goat anti mouse irdye 680, by LI COR (2 mentions)
Sirt1, by Cell Signaling Technology (2 mentions)
Colitis grade dss, by MP Biomedicals (2 mentions)
Antibodies against phosphor total ampk, by Cell Signaling Technology (2 mentions)
Phosphor total erk1 2, by Cell Signaling Technology (2 mentions)
Phosphor total p38, by Cell Signaling Technology (2 mentions)
Phosphor total p65, by Cell Signaling Technology (2 mentions)
Anti ly 6b 2 monoclonal, by Bio-Rad (2 mentions)
Anti β actin antibody, by Bio-Rad (2 mentions)
Dnase 1 and rneasy mini kit, by Qiagen (2 mentions)
104 protocols using vectastain elite abc
1
Quantifying Conjunctival CD4+ T Cells
2
Immunohistochemistry Protocol for Elastin and Fibrillin
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The tissue sections were deparaffinized and hydrated. After blocking the endogenous peroxidase activity, an antigen retrieval step was performed with pepsin at 37°C for one hour. The following primary antibodies were used in this study: a rabbit polyclonal anti-mouse fibrillin 1 (ab21618) (1:200, Abcam, Cambridge, MA, EUA) and an anti-mouse elastin A-19 (SC-17580) (1:600, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibody used for both fibrillin and elastin was VECTASTAIN Elite ABC (Vestor Elite 6105 or PK-PK-6101, Vector Laboratories, Burlingame, CA, USA) and the chromogen used was 3,3'-diaminobenzidine (DAB, Sigma Chemical Co., St. Louis, MO, USA). All sections were then counterstained with Harris’s hematoxylin. Table 1 summarizes the antibodies used for elastin and fibrillin. As negative controls, primary antibodies were omitted from the procedure and bovine serum albumin (BSA) was used instead. The images of elastin and fibrillin were captured and sent to a computer for processing using the optical microscope (Leica DM 4000B, Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) coupled to a digital color camera (Leica DFC295, Leica Microsystems (Switzerland) Ltd, Heerbrugg, Switzerland).
3
Inflammatory Pathway Protein Analysis
4
Histopathological Characterization of Tissues
5
Quantifying Conjunctival CD4+ Cells
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Immunohistochemistry was performed to detect and count the number of cells in the conjunctival epithelium that stained positively for CD4 (clone H129.9, 2 μg/mL; BD Bioscience, San Jose, CA) and anti-rat IgG biotinylated secondary antibody (2 μg/mL goat anti-rat, 559286; BD Pharmingen, San Jose, CA)/Vectastain Elite ABC (using NovaRed reagents; Vector, Burlingame, CA), as previously described.21 (link),22 (link) Secondary antibody alone and proper anti-mouse isotype (BD Biosciences) controls were also performed. Positively stained cells were counted in the goblet cell rich area of the conjunctiva using NIS Elements Software.
6
Immunohistochemical and Immunofluorescence Analysis of Adipose Tissue
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Adipose tissue obtained was fixed in 4% paraformaldehyde overnight, embedded in paraffin, and cut into 4-mm-thick sections. IHC was performed according to the Vecta Stain Elite ABC kit protocols (Vector Laboratories, Burlingame, CA, USA). Antibody staining was visualized with the enhanced DAB kit (Vector Laboratories). The brown reaction product was quantified using Image-Pro Plus software. For IF staining, mature adipocytes were fixed in 4% paraformaldehyde for 15 min, pre-incubated in blocking buffer (10% normal donkey serum in phosphate-buffered saline (PBS)) for 30 min at room temperature, and incubated sequentially with primary and secondary antibodies diluted in blocking buffer. After washing with PBS, the samples were counterstained with DAPI. Cells were placed on slides using cover slips and mounting medium (SouthernBiotech, Burlingame, CA, USA), and examined under a fluorescence microscope (Nikon, Tokyo, Japan).
7
Microglial Immunohistochemistry Protocol
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Rabbit polyclonal antibody against Iba1 (ionized calcium binding adaptor molecule-1) was used as the marker for microglial detection. The protocol was followed as laid down in the Vectastain Elite ABC system kit from Vector Laboratories. Briefly, brain sections were incubated with blocking buffer (1.5% normal goat serum (Vector Laboratories, cat no: S-1000) and 1% Bovine Serum Albumin in 1X PBS) for 2 h at room temperature (RT) followed by incubation with anti-Iba1 antibody, 0.5 μg/ml (Wako Catalog No. 019-19741) in blocking buffer (1.5% normal goat serum (Vector Laboratories, cat no: S-1000) and 1% Bovine Serum Albumin in 1X PBS) overnight at 4°C. The sections were washed with 1X PBS, 3 times and incubated with Biotinylated anti-Rabbit IgG Antibody (1:200) in blocking buffer for 1 h at RT and further washed with 1X PBS, 3 times prior to incubation with Vectastain Elite ABC (Reagent A (1:50) and Reagent B (1:50) in 0.01M PBS) for 1 h at RT. The sections were washed three times and incubated in peroxidase solution (0.01% hydrogen peroxide and 0.05% DAB (3, 3′-diaminobenzidine) HRP substrate in 0.05 M Tris buffer) for color development.
8
Quantification of Splenic Germinal Centers
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A segment of each spleen collected at sacrifice from mice 5–6 months of age was fixed in 10% formalin, processed, embedded in paraffin, and sectioned. After deparaffinization and rehydration, slides were incubated in 0.3% H2O2 for 30 min, washed and incubated for 20 min in 95°C citrate buffer (Vector Laboratories, Burlingame, CA). Slides were blocked for 20 min with Carbo-free blocking solution (Vector Labs), and incubated with a biotinylated anti-PNA antibody (Vector Labs) diluted in phosphate-buffered saline for 30 min. Antibody binding was visualized using Vectastain Elite ABC and DAB reagents (Vector Labs). The number and size of germinal centers based upon PNA staining were quantified using Zeiss Zen Pro software (v4.6.3.0; Carl Zeiss, Thornwood, NY). Quantification was performed by a single observer (J.H.G.) who was blinded to the sex and genotype of the sample.
9
Immunohistochemical Staining Protocol
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FLS were stained with indicated primary antibodies followed by horseradish peroxidase (HRP)-conjugated antibodies and developed by Vectastain Elite ABC and DAB (Vector Laboratories, Peterborough, UK) and then counterstained with Mayer’s haematoxylin. For confocal microscopy, cells and tissues incubated with primary and secondary antibodies and then counterstained with 4,6-diamidino-2-phenylindole.
10
Quantifying Neuronal Subtype Markers in Mouse Hippocampus
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Brains from adult mice (3.5 to 4 months old) were collected and fixed in 4% PFA. Equivalent frozen sections (30 µm thickness) from Dp(16)1Yey (N = 4) and their WT littermates (N = 4) were selected according to common landmarks at positions equivalent to the regions targeted in the in vivo electrophysiology recording experiments (Paxinos and Franklin, 2001 ). Sections were incubated in citrate-EDTA buffer (10 mM citrate, 1 mM EDTA; 80°C for 20 min) and blocked using Avidin/Biotin blocking kit (SP-2001, Vector Laboratories). Sections were then incubated with anti-NPY (1:400, #11976, Cell Signaling Technology; RRID:AB_2716286 ), anti-SST-14 (1:500, T-4103, Peninsula Laboratories; RRID:AB_518614 ) or anti-PV (1:1 000, PC255L, Calbiochem; RRID:AB_2173906 ) and revealed using biotinylated goat anti-rabbit IgG antibody (1:250, Vector BA-1000, Vector Laboratories; RRID:AB_2313606 ) followed by ‘VECTASTAIN Elite ABC’ (PK-6100, Vector Laboratories) and NovaRed substrate kits (SK-4800, Vector Laboratories). Images were acquired using a BZ-X710 light microscope (Keyence) and processed using NIH ImageJ software for particle counting and area calculation. A total of six hippocampal images were counted for each animal in a blinded manner.
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