Hi everyone
I am trying to obtain a good staining for live imaging of microglia on brain slice ( human tissue and mouse)
In particular I am using a lectin reported to stain blood vessels and microglia: DyLight 594 labeled Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), Vectorlabs
I have tryed all the concentration and staining procedures written in literature but it is never comparable at ,for example ,the microglia of CX3CR1 GFP mice. Mainly in terms of penetration of the staining through the tissue.
For this I am really afraid about the quality of my staining in human tissue for which I don't have any good comparison as in mice.
There is someone familiar with these stuff? We can exchange ideas and protocols.
Thank you very much I really appreciate any kind of help
Hi, I definitely agree with you that penetration of the marker is an issue. I was never successful below maybe 80um from the surface... I did find that in my hands, Isolectin B4 (I think from Griffonia simplicifolia) worked better than Tomato Lectin.
Thank you very much for your suggestion. Maybe I will try also this other lectin.
May I ask you tha protocol that you used with Tomato lectin/ Isolectin B4 for the staining? and if it is on rodent slice or human?
Just to have an exchange I am using 30 min of incubation in oxigenated ACFS with 50 micrograms:ml of tomato lectin, wash of 30 min. I noticed that increasing the incubation time to 1 hours doesn't change so much the quality.
Do you have some ideas about that?
really appreciate your help.
Unfortunately I've only used mouse brain slices.
I do the exact same thing as you except I incubate at 32 degrees (mostly to combine recovery and staining, to reduce time from slicing to experiment).
I agree, increasing incubation time just increased background with no appreciable change in signal for me. But perhaps the higher background comes from deeper penetration? I'll test this hypothesis on a confocal sometime soon...
yes it is possible... I have read that maybe filtering the solution in which you have your lectine can decrease the background and increase the quality of the staining... do you do this? in case tomorrow I will try to filter and I let you know
I haven't ever done that but might try it out.. It makes sense if the lectin can aggregate.
Any idea on the filter pore size? 0.45um?
studying the IB4 lectin datasheet i discovered that they claims to filter the solution because lectin gives rise to aggregates.. let's try
I will try with a 0.45, hoping to increase penetration and eliminate some background
Hi..I'm sorry, but I never tried to stain microglia..I always used the endogenous GFP fluorescence in CX3CR1 mice for imaging acquisition.
lectin staining can be species-specific. I know that rat microglia can be stained well with some lectin, but mouse is not with the same one. for me it is an old issue, so i can not help more, sorry..
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