HA was localized using an avidin-biotin-FITC immunofluorescence technique as described with modifications [68 ]. Briefly, the sections were first treated with 1 M glycine to quench endogenous fluorescence. Then, sections were incubated overnight with the biotinylated HA binding region of aggrecan isolated from bovine nasal cartilage (bHABP, Cat# AMS.HKD-BC41, 1:250 dilution; AMS Biotechnology, Cambridge, MA). After washes, bHABP was detected using FITC-conjugated streptavidin (Sigma) and observed microscopically at a magnification of 100×. Specificity of staining for HA was confirmed both by incubation of the probe with excess HA before staining, and by pretreatment of the sections with Streptomyces hyaluronidase to degrade HA before staining (data not shown).
Bhabp
Manufactured by AMS Biotechnology
The BHABP is a laboratory equipment designed for cell culture applications. It functions as a self-contained unit for maintaining stable and controlled environmental conditions essential for cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Ams hkd bc41, by AMS Biotechnology (2 mentions)
Fitc conjugated streptavidin, by Merck Group (2 mentions)
Bhabp, by Merck Group (2 mentions)
K3468, by Agilent Technologies (1 mentions)
Dako antibody diluent, by Agilent Technologies (1 mentions)
Anti ad2 5 e1a antibody, by Santa Cruz Biotechnology (1 mentions)
Accustain trichrome stain kit, by Merck Group (1 mentions)
Dpvr110hrp, by Immunologic (1 mentions)
3 protocols using bhabp
1
Localization of Hyaluronan via Immunofluorescence
2
Localization of Hyaluronan via Immunofluorescence
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HA was localized using an avidin-biotin-FITC immunofluorescence technique as described with modifications [68 ]. Briefly, the sections were first treated with 1 M glycine to quench endogenous fluorescence. Then, sections were incubated overnight with the biotinylated HA binding region of aggrecan isolated from bovine nasal cartilage (bHABP, Cat# AMS.HKD-BC41, 1:250 dilution; AMS Biotechnology, Cambridge, MA). After washes, bHABP was detected using FITC-conjugated streptavidin (Sigma) and observed microscopically at a magnification of 100×. Specificity of staining for HA was confirmed both by incubation of the probe with excess HA before staining, and by pretreatment of the sections with Streptomyces hyaluronidase to degrade HA before staining (data not shown).
3
Immunohistochemical Analysis of E1A, HA, and CD3
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Paraffin-embedded blocks were cut into 4-μm thick sections. E1A and HA staining were performed as previously published,14 (link),21 (link) using the anti-Ad2/5 E1A antibody (1/200; Santa Cruz Biotechnology, SC-25) and B-HABP (5 μg/mL; Amsbio, AMS.HKD-BC41). For human CD3 detection, FLEX Polyclonal Rabbit Anti-Human CD3 (IR503, Agilent DAKO) was diluted 1:10 with DAKO antibody diluent (Dako – Agilent, S0809) for 120 min at room temperature. The secondary antibody used was a BrightVision poly-horseradish peroxidase (HRP)-anti-rabbit immunoglobulin G (IgG) that was biotin-free, ready to use (Immunologic, DPVR-110HRP) incubated 45 min. Antigen-antibody complexes were reveled with 3-3′-diaminobenzidine (K3468, Dako). H&E staining was performed according to standard procedures. Masson trichromic staining was performed using the Accustain Trichrome Stain Kit (Sigma Aldrich) according to the manufacturer’s indications. The percentage of stained areas in IHC images were quantified after color deconvolution by ImageJ FIJI44 (link) software.
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