Confocal Microscopy Method

[admin]

Hi there everyone!

is anyone here an expert on confocal microscopy?

if you have even worked with it once, please post here your experiences.

thanks

Admin

[kmunson779]

You'd probably get more people finding the Confocal forum if its name weren't misspelled.

Confocal microscopy in and of itself is uninteresting. What's interesting is what you do with it, and that's hard to standardize. It's quite different looking at a fixed cell than a live one, for instance, or visualizing protein localization by GFP or immunofluorescence versus localizing chromosomes and genes by FISH, versus measuring nuclear NAD levels by autofluorescence.

I guess if I understood what your goals were for this post, it would make more sense to me.

[admin]

Hey Kmunson,
thanks for spotting that!

Yes, I agree its a general term but I am hoping people with microscopy experience can share their insight into the technique and the methodology of it.

[PeterScherp]

Hi guys,

I would say that I have a good amount of experience with confocal microscopy. I was working with many confocals using many different techniques.
But as it was already posted here: It is better to answer specific questions rather than giving an oversight what you can do with it or how you do it

[cintiadp]

HI there,

confocal microscopy is great if you want to scan a tissue that is thicker than a 6-8um microscope slide. It is great to scan different layers of a tissue, for example, using murine corneas, you can scan from the apical epithelial layers to the basal and even to the stroma cells and even endothelium cells. It all depends on your intention and time. Usually confocal microscopy can be very long.

I hope that helps.

[anjan]

u r right, i do need a help in understanding working with confocal microscopy..interested and experienced lots are invited to post their views..

[playerjs]

I am working in the cell culture area. I am trying to do some cell staining/immunofluorescence by growing cells in cover slips (very problematic - with breakages). I have 2 primary antibodies.. anti-hexokinase 2 and Hsp 60 which is a mitochnodrial marker, we want to conjugate with secondary fluoresecent ab and see if there is any co-localisation.

What advice can you give me interms of a btter or more stringent protocol to ensure I get the images i'm looking for?

1. one important question on my bind is whether or not I should incubate with both primary anibodies simulataneously or in step-wise fashion?

Many thanks

playerJS

[yourlabdata]

I have been working with it for quite some time and use it to visulise FPs in plant tissue. Did you have any specific questions?

I think it is a vital part to my research.