Immunohistochemistry was performed in order to visualize if hyaluronan is present in canine mast cell tumours. Slices of archived paraffin-embedded formalin-fixed CMCT tissue were dewaxed., and endogenous peroxidase blocked by incubation in 1% (v/v) H202 in 70% (v/v) ethanol for 5 min. Sections were washed sequentially in water and PBS and blocked for non-specific binding by a 30 min incubation in 1% (w/v) BSA in PBS. Sections were incubated over night at 4˚C with 2.5μg/ml Biotinylated Hyaluronan Binding Protein (AMS.HKD-BC41, AMSBIO) in 1% (w/v) BSA in PBS. Sections were washed with PBS and incubated with Vectastain Elite ABC Reagent (Vectastain Elite ABC Kit, Vector) for 30 minutes. After an additional wash in PBS the sections were incubated in diaminobenzidine for 7 min. The sections were rinsed in water and counterstained with 10% Mayer’s haematoxylin for 30s. The samples were then washed, dried for two minutes, and mounted in DPX mounting medium.
Ams hkd bc41
Manufactured by AMS Biotechnology
Sourced in United States
The AMS.HKD-BC41 is a laboratory equipment product offered by AMS Biotechnology. It is designed to perform a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Bhabp, by AMS Biotechnology (2 mentions)
Bhabp, by Merck Group (2 mentions)
Fitc conjugated streptavidin, by Merck Group (2 mentions)
Vectastain elite abc reagent, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Streptavidin alexa fluor 568 conjugate, by Thermo Fisher Scientific (1 mentions)
4 protocols using ams hkd bc41
1
Visualizing Hyaluronan in Canine Mast Cell Tumors
2
Localization of Hyaluronan via Immunofluorescence
3
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).
4
Hyaluronan Localization in Cryosections
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Cryosections (15 µm) affixed to glass slides were washed three times with PBS and blocked with 1% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Following a brief rinse with PBS, the tissue sections were incubated at room temperature with a biotinylated recombinant hyaluronan-binding protein (250 µg/mL; AMS.HKD-BC41; AMSBIO, Cambridge, MA, USA) reconstituted in sterile water and diluted (10 µg/mL) in PBS for 1 h. The sections were washed and stained with Streptavidin Alexa Fluor 568 conjugate (2 µg/mL), Wheat Germ Agglutinin Oregon Green™ 488 conjugate (2.5 µg/mL, Thermofisher) and Hoechst 33342 (1 µg/mL, Thermofisher) in PBS for 1 h at room temperature. The slides were mounted with 10% glycerol and stored at 4 °C until imaging.
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