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I am doing immunofluroscence techique and my sample is bone looking for protein localization. I am using the general protocol but its not working i can see staining all over . can any one tell me if im getting the protocol rite my sample is 2x5x5mm
can any one tell me were to upload a pic so that u can guide me better
decalcification- edta
xylene and alcohol washes
antigen retrival -95 degrees using citrate buffer
blocking -h2o2
primary antibody-overnite incubation
secondary antibody-
mounting-waterbased media
im new to this technique. i tried finding protocols but i found it was hard to get one for bone tissue .
im embedding in paraffin. do i need to fix the tissue prior.
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can any one share me teh protocol please..
thanks all
Fri Sep 24, 2010 8:35 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Hi Lotus
What is your protein?
What Ab are you using ? ( please give Supplier number).
What is your secondary Ab conjugated to?
When you state that you use "xylene and alcohol washes " I assume that you have carried out proper dehydration in ascending concentrations of alcohol, clearing in xylene and infiltrating in Pwax before embedding?
Imho, it is very important to FIRST try out your Ab on soft tissue that is identically processed ( but not decalcified) and make sure that you obtain a good signal...then try on your bone sample.
So, we need to know what your protein of interest is, please.
Best wishes,
Carl
Thu Sep 30, 2010 5:21 am
lotus
Rank: Member
Joined: Aug 02, 2010
Posts: 2
Post subject: here are the details
im looking for bone protein : pentosidine
AB primary: antipentosidine monoclonal antibody(cosmobio)
Sec ab: goat anti-mouse Alexafluor 647
washes order
100% xylene
100%etoh 2X
95% etoh 1X
80% etoh 1X
ddwater 2X
we get our sample embedded from outside i dont know before embedding.I fix the tissue decalcify and send them out. what kind of softtissue.Im doing immunofluroscence.
Thu Sep 30, 2010 11:17 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Thanks for data.
If you read the Cosmobio datasheet there are many examples of soft tissues that contain AGE proteins. In the first instance I would use such a tissue as a positive control, to get the antibody optimised.
Why? Because decal may have a deleterious ( bad/negative) effect on your protein making it undetectable.
Also, bone is ALWAYS very hard to carry out immunohistochemistry on and takes a lot of skill/experience to get good results, consistently, imho.
So, get the Ab to work on a soft tissue positive control, then probe the bone.
Also, use another Ab that is known to be positive in your control tissue....for eg: anti Vimentin.
If that works, you know that your technique is good.
How long are you leaving sections in Citric acid?
Also, why use Alexa647?
I would prefer Alexa 594, then I can stain my nuclei with Hoechst or DAPI.
Carl
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