The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 4 µg/ml. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Use a concentration of 5 µg/ml.
1/500 - 1/1000. Detects a band of approximately 22 kDa (predicted molecular weight: 22 kDa).
Plays a major role in tight junction-specific obliteration of the intercellular space.
Note=CLDN4 is located in the Williams-Beuren syndrome (WBS) critical region. WBS results from a hemizygous deletion of several genes on chromosome 7q11.23, thought to arise as a consequence of unequal crossing over between highly homologous low-copy repeat sequences flanking the deleted region.
Ab53156 staining Human colon. Staining is localised to the cell membrane. Left panel: with primary antibody at 4 ug/ml. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffers EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
ICC/IF image of ab53156 stained Mcf7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab53156, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Pasternak JA et al. Claudin-4 undergoes age-dependent change in cellular localization on pig jejunal villous epithelial cells, independent of bacterial colonization. Mediators Inflamm2015:263629 (2015).
Read more (PubMed: 25948883) »
Hwang TL et al. Claudin-4 expression in gastric cancer cells enhances the invasion and is associated with the increased level of matrix metalloproteinase-2 and -9 expression. Oncol Lett8:1367-1371 (2014).
Read more (PubMed: 25120725) »