Hi everyone, I recently purchased an NLRP3 antibody for immunohistochemistry (IHC) from Medchemexpress (HY-P80246), but unfortunately, I haven’t been able to detect any signal in my tissue sections. I’m wondering if anyone has encountered similar issues with NLRP3 detection in IHC. Could it be that this antibody doesn’t work well for IHC? Thank you in advance!
Hi Fan,
Can you give some more information about your staining?
From what organism are your section and how are the embedded?
What was the antibody concentration you used?
Which secondary do you use and which conjugate has the secondary antibody?
The protocol for the staining?
Maybe with some additional information I can help (however I cannot guarantee this - I have made a lot of IHC but not worked with this antibody specifically)
Hello Fan Janice
It cannot happen that NLRP3 antibody for immunohistochemistry will not work well for IHC. Have you used a positive control? If the positive control also shows no signal, it could indicate a problem with the antibody or the detection system.
No signal detection in IHC can stem from several issues.
1. Improper fixation can mask or degrade the target antigen, preventing antibody binding.
2. Incomplete removal of paraffin could prevent antibody binding.
3. Insufficient antigen retrieval could prevent antibody access to the target antigen.
4. If the sections that are too thick or too thin, it can affect signal detection.
5. What type of detection system are you using? If you are using a biotin-based detection system, inadequate blocking of endogenous biotin can lead to high background and mask the signal.
6. There can be issues with the detection system as well, such as enzyme activity or substrate reaction, can prevent proper signal generation.
Re-visit your protocol and work on the above suggestions. Maybe you might succeed.
Best,
Dörthe Andrea Kesper Thank you for your reply and for offering to help. Here are the details of my staining protocol:
Tissue source: The sections are from mouse tissue.
Embedding method: The samples were fixed in 4% paraformaldehyde and embedded in paraffin. Sections are 5 µm thick.
Antigen retrieval: I used citrate buffer (pH 6.0) with heat-induced epitope retrieval (microwave, 15 min).
Blocking: I used 5% normal goat serum for 1 hour at room temperature.
Primary antibody: NLRP3 antibody from Medchemexpress (HY-P80246), used at 1:100 dilution overnight at 4°C.
Secondary antibody: Goat anti-rabbit IgG (H+L) conjugated to HRP (from Cell Signaling Technology), incubated for 1 hour at room temperature.
Detection: DAB chromogen for signal development, counterstained with hematoxylin.
Hi Fan,
I think you got all lot of good recommendations from Malcolm Nobre and for me your protocol sounds ok. I don't know how you deparaffinzed and dehydrated the sections but I would conclude this was made with xylene and alcohol series what should be ok.
I would also think that the thickness of the sections is ok.
However, regarding your retrieval buffer: you use a citrate buffer with pH6 (which is often used), but the efficacy of antigen retrieval can be really influenced by the pH of the buffer and it seems that a pH of 9 is often better working.
I found a protocol for the NLRP3 antibody from Proteintech were they used a pH9 Tris EDTA buffer (https://www.ptglab.com/protocol/27458-1-APIHC.pdf).
You might also check out this publication: "Mechanisms of Heat-induced Antigen Retrieval: Does pH or Ionic Strength of the Solution Play a Role for Refolding Antigens?" - you will find the full text on research gate.
So I would try the ph9 Tris EDTA puffer for antigen retrival.
And as Malcom mentioned an positive control (if you have one) would be a good idea to see if every thing else is working.
I hope this helps.
KR Dörthe
The low expression level of NLRP3 in normal or non-activated tissues/cells is a major factor—for example, in mouse liver and kidney tissues. Therefore, it is necessary to stimulate cells with agents such as LPS to induce NLRP3 expression. Before detection, the expression level of the target protein should be confirmed, and a positive control should be included in the experiment. For instance, whole cell lysate from RAW 264.7 cells treated with 10 µg/mL lipopolysaccharide (LPS) for 8 hours can be used as a positive control.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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