After single cell or paired-recordings, slices containing biocytin-filled neurons were fixed for at least 24 h at 4°C in 100 mM phosphate buffer solution (PBS, pH 7.4) containing 4% paraformaldehyde (PFA). After several rinses in PBS, slices were treated with 1% H2O2 in PBS for approximately 20 min, in order to quench endogenous peroxidase activity. Following this, slices were rinsed several times using 100 mM PBS and subsequently incubated in 1% avidin-biotinylated horseradish peroxidase (Vector ABC staining kit, Vector Lab. Inc.) containing 0.1% Triton X-100 for 1 h at room temperature. This was followed by a chromogenic reaction that resulted in a dark precipitate by adding 0.5 mg/ml 3,3-diaminobenzidine (DAB; Sigma-Aldrich) until distinct axonal and dendritic branches of the biocytin-filled neurons were clearly visible. The slices were rinsed again with 100 mM PBS, followed by slow dehydration in increasing ethanol concentrations and finally in xylene for 2–4 h. Slices were then mounted on gelatinized slides and embedded using Eukitt medium (Otto Kindler GmbH).
Vector abc staining kit
Manufactured by Vector Laboratories
Sourced in United States
The Vector ABC staining kit is a laboratory product designed for immunohistochemical staining. It provides a consistent and reliable method for the detection of target antigens in fixed tissue samples.
Automatically generated - may contain errors
8 protocols using vector abc staining kit
1
Biocytin-Filled Neuron Visualization
2
Biocytin-Filled Neuron Visualization
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After the electrophysiological recordings, slices containing biocytin-filled neurons were processed using a protocol described previously (Marx et al. 2012 (link); Radnikow et al. 2012 ). The slices were fixed in 4% PFA (12.9 mM) in 100 mM phosphate buffer solution (PBS) for at least 24 h at 4 °C. To block any endogenous peroxidase activity, the slices were treated with 3% H2O2 (29.4 mM) solution in PBS for about 20 min. The slices were rinsed repeatedly using 100 mM PBS and subsequently incubated at room temperature in 1% avidin-biotinylated horseradish peroxidase (Vector ABC staining kit, Vector Lab. Inc.) containing 0.1% Triton X-100 for 1 h. This was followed by chromogenic reaction by adding 0.5 mg/ml (13.4 mM) 3,3-diaminobenzidine (DAB; Sigma-Aldrich, USA) until the biocytin-filled neurons with distinct axonal and dendritic branches were clearly visible. The slices were rinsed again with 100 mM PBS, then dehydrated slowly for 2–4 h with increasing ethanol concentrations and finally in xylene (see Marx et al. 2012 (link) for details). The slices were then mounted on gelatinized slides and embedded using Eukitt medium (Otto Kindler GmbH).
3
Biocytin-Filled Neuron Visualization
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Slices were fixed after electrophysiological recordings with 4% paraformaldehyde in 100 mM phosphate buffered saline (PBS) for at least 24 h at 4°C. To recover the morphology of biocytin-filled neurons, slices were rinsed several times in 100 mM PBS and then treated with 1% H2O2 in PBS for about 20 min in order to reduce any endogenous peroxidase activity. Slices were rinsed repeatedly with PBS and then incubated in 1% avidin-biotinylated horseradish peroxidase (Vector ABC staining kit, Vector Lab. Inc., Burlingame, CA, United States) containing 0.1% Triton X-100 for 1 h at room temperature. The reaction was catalysed using 0.5 mg/ml 3,3-diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO, United States) as a chromogen. Slices were then rinsed with 100 mM PBS, followed by slow dehydration with ethanol in increasing concentrations and finally in xylene for 2–4 h. After that, slices were embedded using Eukitt medium (Otto Kindler GmbH, Freiburg, Germany).
4
Biocytin Neuron Visualization Protocol
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After the electrophysiological recordings, slices containing biocytin-filled neurons were fixed for at least 24 h at 4 °C in 100 mM phosphate buffer solution (PBS, pH 7.4) containing 4% paraformaldehyde (PFA). After rinsing slices several times in PBS, they were treated with 1% H2O2 in PBS for about 20 min, in order to reduce any endogenous peroxidase activity. Following this, slices were rinsed repeatedly using 100 mM PBS, then incubated in 1% avidin-biotinylated horseradish peroxidase (Vector ABC staining kit, Vector Lab. Inc.) containing 0.1% Triton X-100 for 1 h at room temperature. This was followed by a chromogenic reaction that resulted in a dark precipitate by adding 0.5 mg/ml 3,3-diaminobenzidine (DAB; Sigma-Aldrich, USA) until distinct axonal and dendritic branches of the biocytin-filled neurons were clearly visible. The slices were rinsed again with 100 mM PBS, followed by slow dehydration in increasing ethanol concentrations and finally in xylene for 2–4 h (Marx et al. 2012 (link)). Slices were then mounted on gelatinized slides and embedded using Eukitt medium (Otto Kindler GmbH).
5
Immunohistochemical Analysis of Mouse Skin
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The samples of imiquimod-applied mouse back skin were harvested. They were formalin-fixed and stained with hematoxylin and eosin. For immunohistochemistry, the mouse skin was embedded in an optimal cutting compound, snap-frozen in liquid nitrogen and stored at −80 °C. Cryosections were fixed with cold acetone for 5 min and incubated overnight at 4 °C with anti-mouse MHC class II antibody (1/50 dilution, Abcam, Cambridge, UK) and anti-mouse CD3 antibody (1/100 dilution, Abcam). Tissues were subsequently stained with an avidin-biotin peroxidase complex using a Vector ABC staining kit (Vector Laboratories, Burlingame, CA). Diaminobenzidine was used for visualizing the staining, and counterstaining with Mayer hematoxylin was performed according to the manufacturers’ instructions. Stained cells were counted in 10 random grids under high original magnification (×400) power fields of a light microscope. Each section was examined independently by two investigators in a blinded manner. In some experiments, 5 μm-thick tissue sections from freshly frozen samples were stained with fluorescein isothiocyanate (FITC)-conjugated IL-17A (BioLegend, San Diego, CA, USA) and FITC-conjugated IFN-α (Biorbyt, Cambridge, UK), or FITC-conjugated IL-10 (BioLegend).
6
Immunohistochemical Analysis of Mouse Skin
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Samples of imiquimod-applied mouse back skin were harvested. They were formalin-fixed and stained with hematoxylin and eosin. For immunohistochemistry, mouse skin was snap-frozen and stored at –80 °C. Cryosections were fixed with cold acetone for 5 min and incubated overnight at 4 °C with anti-mouse major histocompatibility complex class II antibody (1/50 dilution, Abcam, Cambridge, UK), anti-mouse CD3 antibody (1/100 dilution, Abcam), anti-mouse Gr-1 antibody (1/100 dilution, BD Pharmingen, San Diego, CA) or anti-mouse F4/80 antibody (1/100 dilution, Bio-Rad, Hercules, CA). Tissues were subsequently stained with an avidin-biotin peroxidase complex using a Vector ABC staining kit (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine was used for visualizing the staining, and counterstaining with Mayer hematoxylin was performed according to the manufacturers’ instructions. Stained cells were counted in 10 random grids under high original magnification (×400) power fields of a light microscope. Each section was examined independently by two investigators in a blinded manner.
7
Biocytin-Filled Neuron Visualization
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After the electrophysiological recordings, slices containing biocytin-filled neurons were fixed at 4 °C in 100 mM phosphate buffer solution (PBS, pH 7.4) containing 4% paraformaldehyde (PFA) for at least 24 h. After rinsing slice several times in PBS, slices were treated with 1% H 2 O 2 in PBS for about 20 min, in order to reduce any endogenous peroxidase activity. Following this slices were rinsed repeatedly using 100 mM PBS, then incubated in 1% avidin-biotinylated horseradish peroxidase (Vector ABC staining kit, Vector Lab. Inc.) containing 0.1% Triton X-100 for 1 h at room temperature. This was followed by a chromogenic reaction that resulted in a dark precipitate by adding 0.5 mg/ml 3,3-diaminobenzidine (DAB; Sigma-Aldrich, USA) until distinct axonal and dendritic branches of the biocytin-filled neurons were clearly visible. The slices were rinsed again with 100 mM PBS, followed by slow dehydration in increasing ethanol concentrations and finally in xylene for 2-4 h (Marx et al. 2012) . Slices were then mounted on gelatinized slides and embedded using Eukitt medium (Otto Kindler GmbH).
8
Biocytin-Filled Neuron Visualization
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Slices were fixed after electrophysiological recordings with 4% paraformaldehyde (PFA) in 100 mM phosphate buffered saline (PBS) for at least 24 h at 4°C. To recover the morphology of biocytin-filled neurons, slices were rinsed several times in 100 mM PBS and then treated with 1% H2O2 in PBS for about 20 min in order to reduce any endogenous peroxidase activity. Slices were rinsed repeatedly with PBS and then incubated in 1% avidin-biotinylated horseradish peroxidase (Vector ABC staining kit, Vector Lab. Inc., Burlingame, USA) containing 0.1% Triton X-100 for 1 h at room temperature. The reaction was catalysed using 0.5 mg/ml 3,3-diaminobenzidine (DAB; Sigma-Aldrich, St.Louis, Mo, USA) as a chromogen. Slices were then rinsed with 100 mM PBS, followed by slow dehydration with ethanol in increasing concentrations and finally in xylene for 2-4 h. After that, slices were embedded using Eukitt medium (Otto Kindler GmbH, Freiburg, Germany).
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