I am working with plant tissues, buds specifically, fixed in glutaraldehyde and embedded in LR White resin. I am working with the primary antibodies LM19 and LM20 targeting pectins. I am using a secondary antibody conjugated with FITC, and regardless of using the proper configuration in the confocal microscope (LSM710), I can barely see the stain. I tried linear unmixing with a similar result. I can barely see the stain because there is a high amount of autofluorescence (due to glutaraldehyde fixation).
I wondered if it is valid to use a different type of reporter, as HRP or ALP. However, I am not sure if this is acceptable for something different than localization of compounds. Are there other pigments conjugated to secondary antibodies instead of using fluorophores (and then not deal with the autofluorescence)?
I will appreciate any information. Thanks!
Can you try another secondary in the red channel like Texas red? Autofluorecence bleeds most into the fitc channel, as well, it photobleaches quite quickly compared to other fluorchromes
Hi Camilo,
You cumulate difficulties : plant material, glutaraldehyde fixation, LR White resin.
As you fixed with GA you generated high autofluorescence, and as it is embedded in LRWhite, autofluorescence is also embedded. Moreover, with LR resins, only epitope at the surface of the cut are vailable to antibody binding, thus you have very few antibodies bound, leading to a weak signal. LR white is rather used for immunoGold for TEM.
For immunofluorescence on plant tissue you should use material fixed with formaldehyde, embedded in Steedman's wax, cut 10-20µm thick, and use in your immuno buffer 1% BSA (or gelatin) and 10 mM Na2SO5 in order to reduce autofluorescence.
If you have gold conjugated secondary antibodies for TEM you can turn the problem, by imaging the autofuorescence as background, and gold reflection of the laser as signal.
Cheers,
Oliv
I'm not sure that HRP will be OK, because the fluorecent products are soluble, and will move by diffusion, and laser scanning will favor this diffusion by local heating.
You can try Tyramide amplification, it worked quite well for me some years ago on root samples.
Another possibility is to use AlloPhycoCyanin (APC) or PhycoErythrin (PE) coupled secondary antibodies used for flow cytometry. PE and APC are huge fluorescent proteins that emit in light-red (PE) or deep-red (APC) with broad excitation spectrum and narrow emission. They are multimeric and have high quantum yield. Thus for 1 primary IgG, you can have 12 fluorochromes, exhibiting high fluorecence.
Hi
Lora Benoit Thank you for your recommendation. I explored the autofluorescence with lasers with longer wv (594 and 633nm) and will try using a secondary in the red channel. I am still deciding between Alexa Fluor 594 or Alexa Fluor 633.
Olivier Catrice Thank you for your recommendations too. Yes, as I am working more with microscopy I've been learning the difficulties of fixing with GAA and embedding with LR white resin. Thank you for helping me discard the idea of using HRP.
Ma Junwei The truth I don't know the reason why Na2SO5 could reduce autofluorescence. Maybe Olivier Catrice could have an idea.
Na2S2O5 is a reducing agent that can reduce some compounds such as tannins and other polyphenolics, flavonoids ... and then reduce the autofluorescence. Many reducers will function such as H3BO4, Mercaptoethanol, DTT ... But DTT and mercaptoethanol are volatiles and fumes are toxic, boric acid, while non-volatile, is a CMR. Na2S2O5 is safe, cheap and colorless.
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