Three brain sections per animal were selected to contain the ventral posteromedial (VPM)/ventral posterolateral (VPL) nuclei of the thalamus and the somatosensory barrel field (S1BF) cortex. Immunohistochemistry was performed as previously described66 (link). Briefly, free floating brain sections were incubated in 1% hydrogen peroxide in Tris-buffered saline (TBS) for 20 min to block endogenous peroxidase activity followed by 3 washes in TBS. Sections were then blocked in 15% goat serum-containing TBS-T (TBS with 0.3% Triton X-100) for 30 min followed by primary antibody solution overnight at 4 °C. Primary antibodies included: anti-CD68 (BioRad AbD Serotec, MCA1957; 1:2,000), anti-GFAP (Dako, Z0334; 1:8,000), and anti-ATP synthase C (Abcam, ab181243, 1:1,000) diluted in TBS-T+ 10% goat serum. Secondary antibodies included: biotinylated anti-rat and anti-rabbit IgGs (Vector Labs, BA-9400, BA-1000; 1:2000) diluted in TBS-T+ 10% goat serum. An ABC amplification kit (Vector Labs) was used for 2 h followed by 0.05% DAB solution until visible reaction had occurred.
Abc amplification kit
Manufactured by Vector Laboratories
Sourced in United States
The ABC amplification kit is a laboratory tool used for the amplification of nucleic acid sequences. It provides a set of reagents and protocols for the efficient and reliable amplification of DNA or RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Z0334, by Agilent Technologies (1 mentions)
Ab181243, by Abcam (1 mentions)
Mca1957, by Agilent Technologies (1 mentions)
Bright field microscope, by Zeiss (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Axio imager d1, by Zeiss (1 mentions)
2 protocols using abc amplification kit
1
Immunohistochemical Analysis of Brain Regions
2
Multimodal Vascular Tissue Characterization
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Ten mm thick OCT embedded frozen cross sections were stained with Orcein and Sirius red for visualisation of elastic and collagen fibers, respectively. Immunostaining on OCT embedded frozen cross sections was performed using the following antibodies: mouse monoclonal anti-aSMA actin (clone 1A4, Sigma, Lyon, France), mouse anti-PCNA (Proliferative cell nuclear antigen, clone PC10, SigmaeAldrich, St Louis, WA, USA). After incubation with a biotin conjugated anti-species antibody, immunostaining was amplified using an ABC amplification kit (Vector Laboratories, Burlingame, CA, USA). Peroxidase was detected using DAB (Vector Laboratories). For thrombus detection, cross sections were incubated with rabbit HRP-conjugated anti-fibrinogen antibody (Dako, Glostrup, Denmark) and DAB. Sections were counterstained with haematoxylin, mounted in Eukitt and examined with a bright field microscope (Zeiss, France). For immuno-fluorescence analysis, 10 mm thick frozen cross sections were incubated with the mouse anti-SMA-Cy3 antibody (clone 1A4, SigmaeAldrich, USA). Nuclei were stained with DAPI and sections were mounted in Mowiol. Fluorescence was examined with a fluorescence microscope (AxioImager D1, Zeiss) in sequential scanning mode for quadruple detection of Cy3, DAPI, PKH26, and elastin green auto fluorescence.
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