I am using the Chemicon Polyclonal Rabbit anti-mouse Collagen IV on mouse kidney samples that have been fixed in 4% paraformaldehyde, processed rountinely and embedded in paraffin. I cut the sections at 3 microns and ran a routine IHC protocol using Proteinase K (DAKO, ready-to-use, 10mins) as a retreival method and DAKO Envision (HRP) kit as secondary.
Is anyone familar with Collagen IV staining pattern within the kidney??
At the moment for all my samples I am receiving areas of weak staining in the tubules closest to the outside edge of the kidney. Otherwise, the remaining basement membranes and areas within the glomeruli are stained very nicely (dark DAB staining). The faint staining appears in patches and I cannot tell if it is artifact (over digestion with Prot. K), a technical error or an actually pathological phenomena. I ran two batches, one baked the day of staining at 65 degree C and another overnight at 37 degrees C thinking that maybe baking at high temps damaged the epitope. But the pattern I received is the same for both batches.
Any information would be helpful. I am just not familar with the "normal" pattern that collegen IV stains within the kidney.
Thank you very much.
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