Anti-Hsp60 抗体 (ab46798)

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ab46798 staining Hsp60 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab46798 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

IHC image of Hsp60 staining in a section of formalin-fixed paraffin-embedded normal human liver* performed on a Leica Biosystems BOND® RX instrument. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab46798, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Blocking Buffer: 5% NFDM/TBST

Dilution Buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human lung adenocarcinoma with purified ab46798 at a working dilution of 1 in 100. The secondary antibody used is ab97051 Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Overlay histogram showing HeLa cells fixed in 2% PFA and stained with purified ab46798 at a dilution of 1 in 150 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 150. Rabbit monoclonal IgG was used as an isotype control (black) and cells without incubation with the antibody were used as a negative control (blue line).

ab46798 (purified) at 1/40 immunoprecipitating Hsp60 in MCF7 (Lane 1). Lane 2 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Blocking Buffer: 5% NFDM/TBST

Dilution Buffer: 5% NFDM/TBST

Blocking Buffer: 5% NFDM/TBST

Dilution Buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded rat kidney with purified ab46798 at a working dilution of 1 in 100. The secondary antibody used is ab97051 Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin embedded mouse cardiac muscle with purified ab46798 at a working dilution of 1 in 100. The secondary antibody used is ab97051 Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin embedded human cervical carcinoma with purified ab46798 at a working dilution of 1 in 100. The secondary antibody used is ab97051 Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Unpurified ab46798 staining Hsp60 from human colon tissue by immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). Cells were formaldehyde fixed prior to blocking in 10% serum for 2 hours at 21°C. The primary antibody was diluted 1/500 and incubated with the sample for 2 hours at 21°C. Alexa fluor® 594 goat polyclonal, diluted 1/5000, was used as the secondary.

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Immunohistochemical analysis of paraffin-embedded human breast adenocarcinoma using unpurified ab46798 at 1/250 dilution.

Blocking Buffer: 5% NFDM/TBST

Dilution Buffer: 5% NFDM/TBST