Tangential sections were taken to include islet and renal tissue. Formaldehyde-processed specimens were stained with hematoxylin and eosin (H&E) and insulin-specific antibodies. Insulin staining was performed on 4.0-µm sections. Sections were incubated at room temperature in a humidified chamber for 20 min with 10% normal goat serum in phosphate buffered saline (PBS; pH 7.4) and then for 60 min with guinea pig antiporcine insulin antibody (Dako, Carpinteria, CA, USA) diluted 1:10 in PBS. They were next incubated with a 1:200 dilution of biotinylated goat antiguinea pig secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 60 min. The tissue-bound primary antibodies were detected by an avidin–biotin–peroxidase complex (Dako) that was visualized by staining with 0.02% hydrogen peroxide containing 0.3 mg/mL 3,3′-diaminobenzidine (DAB) in 0.05 mol/L Tris buffer. Sections were counterstained with Gill’s single-strength hematoxylin.
Biotinylated goat antiguinea pig secondary antibody
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated goat antiguinea pig secondary antibody is a laboratory reagent used for the detection and identification of guinea pig primary antibodies in various immunoassays and immunohistochemical techniques. It provides a means to amplify the signal generated by the primary antibody-antigen interaction.
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2 protocols using biotinylated goat antiguinea pig secondary antibody
1
Immunohistochemical Analysis of Insulin in Tissues
2
Immunohistochemical detection of c-Fos in brain sections
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Brain sections were rinsed in 0.1M PBS with 3% H2O2. Then, brain sections were incubated for 1 h in a blocking solution of 0.1M PBS containing 3% normal goat serum and 0.1% triton X-100, followed by incubation with the primary antibody polyclonal guinea pig anti-c-Fos (Synaptic Systems, ref 226 004, 1:1000) at 4 °C for 72 h. All steps following incubation with the primary antibody were performed at room temperature (RT).
Next, brain sections were rinsed in 0.1M PBS-T and incubated with biotinylated goat anti-guinea pig secondary antibody (Vector, ref BA-7000-1.5, 1:500) for 1 h at RT. Samples were washed in PBS and incubated with VECTASTAIN® Elite® ABC HRP (PK-6100, Vector, A reagent 1:250 and B reagent 1:250) in PBS for 1 h at RT. Tissues were rinsed with PBS and then 0.05 M Tris buffer. Revelation was performed in 0.003% H2O2 with 0.05% 3,3′-diaminobenzidine (DAB) in 0.05 M Tris buffer for 11 min. Frozen sections were dehydrated in an ethanol series at 50%, 70%, 90%, and 100% and SaveSolv (VWR Q-Path) for 10 min each. Finally, the sections were mounted onto glass slides with Eukitt mounting medium (Sigma Aldrich) and coverslipped.
Next, brain sections were rinsed in 0.1M PBS-T and incubated with biotinylated goat anti-guinea pig secondary antibody (Vector, ref BA-7000-1.5, 1:500) for 1 h at RT. Samples were washed in PBS and incubated with VECTASTAIN® Elite® ABC HRP (PK-6100, Vector, A reagent 1:250 and B reagent 1:250) in PBS for 1 h at RT. Tissues were rinsed with PBS and then 0.05 M Tris buffer. Revelation was performed in 0.003% H2O2 with 0.05% 3,3′-diaminobenzidine (DAB) in 0.05 M Tris buffer for 11 min. Frozen sections were dehydrated in an ethanol series at 50%, 70%, 90%, and 100% and SaveSolv (VWR Q-Path) for 10 min each. Finally, the sections were mounted onto glass slides with Eukitt mounting medium (Sigma Aldrich) and coverslipped.
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