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.
Novared reagents
Manufactured by Vector Laboratories
Sourced in United States
NovaRed reagents are a product offered by Vector Laboratories. They are used as a chromogenic substrate for the detection of peroxidase enzyme activity in immunohistochemistry, immunocytochemistry, and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain elite abc, by Vector Laboratories (3 mentions)
Biotinylated secondary antibody, by BD (1 mentions)
Nis elements software, by Nikon (1 mentions)
Cd4 clone h129, by BD (1 mentions)
Anti mouse isotype, by BD (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Rodent block m, by Biocare Medical (1 mentions)
Rodent decloaker, by Biocare Medical (1 mentions)
Vulcan fast red chromogen kit 2, by Biocare Medical (1 mentions)
Abc complex, by Vector Laboratories (1 mentions)
Eclipse 50i, by Nikon (1 mentions)
Nis elements version 4, by Nikon (1 mentions)
Goat anti rat antibody, by BD (1 mentions)
Rat anti mouse cd4 antibody, by BD (1 mentions)
5 protocols using novared reagents
1
Quantifying Conjunctival CD4+ T Cells
2
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.
3
Neutrophil Quantification in Cornea
4
Immunohistochemical Analysis of Tissue Samples
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Immunostaining and Congo red histochemistry were performed on 5 μm paraffin sections. Antigens were retrieved in 25 mM citrate buffer (pH 7.2) or Rodent Decloaker (Biocare Medical, USA) using a microwave oven. Sections were blocked with M.O.M.™ IgG block (Vector Labs, USA) or Rodent Block M (Biocare Medical). Primary antibody (Additional file 1 : Table 1) incubation was carried out overnight at ~ 4 °C followed by incubation with secondary antibody for 30–60 min at room temperature. ABC™ complex and NOVA™ red reagents (Vector Labs) were used to visualize the immunosignals. In other settings, MM AP-Polymer kit (mouse antibody on mouse tissues) or MACH3™ Rabbit-Probe Alk Phos Polymer kits were employed along with Vulcan Fast Red Chromogen kit 2 (Biocare Medical). For double immunostaining using fluorescent secondary antibodies, primary antibodies were incubated overnight, simultaneously or stepwise at 4 °C, followed by incubation with the appropriate secondary antibodies (Alexa Fluor, fluorescent secondary antibodies against mouse and rabbit, Invitrogen). DAPI (4′,6-diamidino-2-phenylindole) was used for counterstaining of nuclei. Sulfated alcian blue staining was performed according to Lendrum et al. [28 (link)].
5
Conjunctival CD4+ Cell Quantification
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Immunohistochemistry was performed to detect and count the cells in the conjunctiva that stained positively for CD4. Cryosections were stained with primary rat anti-mouse CD4 antibody (1:50; catalog no. 553647; BD Pharmingen, San Diego, CA, USA), goat anti-rat antibody (1:25; catalog no. 559286; BD Pharmingen) and Vectastain Elite ABC using NovaRed reagents (catalog no. PK-6100; Vector). Secondary antibody alone and appropriate anti-mouse isotype (BD Pharmingen) controls were also performed. Three slides from each animal were examined and photographed with a microscope equipped with a digital camera (Eclipse 50i; Nikon, Tokyo, Japan). Positively stained cells were counted in the conjunctiva using image analysis software (NIS Elements, version 4.1, Nikon, Melville, NY, USA).
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