Rabbit specific hrp dab abc detection ihc kit

Brains were fixed in 4% paraformaldehyde at 4 °C for 48 h before dehydration and embedding in paraffin. Next, 5 μM thick coronal sections were cut with a microtome and mounted on glass slides. The sections were deparaffinised in xylene and rehydrated through decreasing concentrations of ethanol. Dopaminergic neurons were labeled with TH. The immunoreactivity of TH was visualized using the Rabbit-specific HRP/DAB (ABC) Detection IHC Kit (ab64261, Abcam, VIC, Australia) according to the manufacturer’s protocol. Negative controls were created for all regions analysed by not incubating with the primary antibody. Hematoxylin was used as a counterstain to visualize nuclei. Sections were dehydrated in increasing concentrations of ethanol and xylene before being mounted. Images were taken on a ZEISS AxioScan.Z1 (Carl Zeiss Australasia, NSW, Australia) at ×20 magnification. Fiji ImageJ was used for the semi-quantification of DAB-positive cells normalized to nuclei (hematoxylin stain) [53 (link)].

Breast tumors and lungs fixed in 10% formalin were embedded in paraffin and sectioned. The hydrated slides were microwaved in a Tender Cooker (Nordic Ware, Minneapolis, MN, USA) for 30 min in 10 mM citric acid (pH 6.0) for antigen retrieval. A Rabbit-specific HRP/DAB (ABC) Detection IHC Kit (Abcam, Toronto, ON, Canada) was used according to the manufacturer’s instructions. The rabbit anti-CD31 (ab28364, 1:80) antibody was from Abcam (Toronto, ON, Canada), and the rabbit anti-Ki67 (D3B5, 1:200) was from Cell Signaling Technology (Whitby, ON, Canada). Positive staining events for CD31 and Ki67 were selected and counted using ImageJ. An analysis was made using images taken with a 5× magnification lens, and the illustration was taken with a 20× magnification lens. CD31 was quantified as the number of vessels per field, and Ki67 as a percentage of the positive cells. The results were averaged from 10 image fields for each sample.

For immunohistochemistry we used an immunohistochemical staining method previously utilized to immunolocalize the MMTV-like p14 protein in human breast cancer [19 (link)] and validate by FISH analysis [20 ]. Four-micron thick sections were dewaxed in xylene and rehydrated through graded alcohols to water. Antigen retrieval was performed microwaving sections for 9 minutes in citrate/EDTA buffer (pH 7.8). Not specific peroxidase activity was blocked with 3% hydrogen peroxidase for 15 minutes, and non-specific binding prevented by incubation with normal goat serum for 10 minutes. Afterwards, incubation with 1:2000-diluted rabbit polyclonal antibody anti MMTV-p14 (kindly provided by Dr J Hochman, University of Jerusalrm, Israel) was performed for 2 hour at room temperature. Negative controls included the omission of the primary antibody. A biotin conjugated goat derived secondary antibody was applied followed by the enzyme-labeled streptavidin and substrate chromogen (Rabbit specific HRP/DAB-ABC detection IHC kit, ab 64261 abcam). Slides were counterstained with hematoxylin. Cytoplasmic/nuclear staining for MMTV-p14 were considered positivity.

Cleaved PARP primary antibody (Abcam Cat# ab32064, RRID:AB_777102) was used at a 1:6000 dilution and the rabbit-specific HRP/DAB (ABC) detection IHC kit (Abcam) was used for immunohistochemistry, according to the manufacturer’s instructions. Sections were counterstained with haematoxylin and mounted using Vectamount permanent mounting media (Vector Labs, Peterborough, United Kingdom). Images were taken at 40x magnification on a Hamamatsu Nanozoomer Digital slide scanner. Cleaved PARP-positive cells were scored as the percentage of cells with nuclear staining.