Immunohistochemical analysis of formalin fixed paraffin embedded mouse small intestine labelling GSDMD with ab219800 at a concentration of 0.05µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab219800 anti-GSDMD antibody [EPR20859] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse small intestine labelling GSDMD with ab219800 at a concentration of 0.1µg/ml. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20mins.

ab219800 anti-GSDMD antibody [EPR20859] was incubated for 15mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labelling GSDMD with ab219800 at 1/1000 (0.542 μg/ml) followed by ab214880 at a ready to use dilution. Low expression tissue: weak staining on mouse lung. The section was incubated with ab219800 at 4°C overnight. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is ab214880 at a ready to use dilution.

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Exposure time : Lanes 1 and 2:3 minutes; Lanes 3 and 4:48 seconds; Lanes 5 and 6:3 minutes.

Blocking/Dilution buffer: 5% NFDM/TBST.

Expression level of GSDMD in whole normal brain lysate is low or undetectable (PMID: 32671214, PMID: 34975487), we recommend loading higher amount of lysate or using lower antibody dilution.

Exposure time: Lanes 1 and 2:15 seconds; Lanes 3 and 4:37 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

The antibody has strong reactivity with GSDMD with a few weak bands of undermined proteins.

The lysates were kindly provided by our collaborator Dr Feng Shao's lab, NIBS.

Immunohistochemical analysis of paraffin-embedded Wild type mouse small intestine (A) and GSDMD KO mouse small intestine (B) tissue labeling GSDMD with ab219800 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on epithelium of wild type mouse small intestine (A), no staining on GSDMD KO mouse small intestine (B) (PMID: 17350798) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

The tissue samples were kindly provided by Dr. Feng Shao NIBS.

Heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 was performed before commencing with IHC staining protocol.

Blocking and diluting buffer: 5% NFDM /TBST

Flow cytometry overlay histogram showing Raw264.7 cells stained with ab219800 (red line). The cells were fixed with 80% Methanol and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10µg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab219800) (1x 10⁶ in 100µl at 0.2µg/ml (1/2)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

GSDMD was immunoprecipitated from 0.35 mg of RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate with ab219800 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab219800 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.