[Histonet] Re: Autofluorescence/Imunofluorescence protocols rat kidney

[c.thompson <@t> student.ucc.ie]

Hi Histonet,

A very basic question but I am a complete novice in the
world of immunohistochemistry / immunofluorescence and am
trying to teach myself the rudiments!

At present, I am attempting to visualise NHE3 in rat kidney
using immunofluorescence but am encountering a lot of
autofluorescence.  I just wanted to enquire about possible
reasons for / methods of dealing with this.

My method is as follows:

Once the kidneys have been excised I fix them in 4%
paraformaldehyde overnight (4 degrees celsius) and then
incubate them in 30% sucrose until they sink. Kidneys are
then frozen in isopentane in liquid nitrogen and
cryosectioned.

Sections rinsed in distilled water
3 X 5 min washes in PBS
Incubation in blocking solution (3% normal goat serum, 0.25%
Triton X. 96.75% PBS) for 1 hr @ room temp
Slides then incubated with primary antibody (1:500 chemicon
anti nhe3) in a humidified chamber @ 4  degrees overnight

Slides rinsed in distilled water 3 X 5 min washes in PBS
Incubation with 1:500 FITC conjugated secondary antibody for
1 hr at room temp
slides rinsed and coverslips applied with vectashield,
sealed and visualised

Apologies for all the detail, just generally unsure of my
method... and whether there are more apt methodologies for
this? Getting a lot of autofluorescence and finding it quite
difficult to visualise the NHE3 at all!

Would really appreciate any help at all!

Thank you, 
Claire