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Author Topic: please check my protocol for me!  (Read 6968 times)

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Offline ASluo

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please check my protocol for me!
« on: November 07, 2008, 12:47:11 AM »
Hi guys,

Please have a look at the my protocol and let me know if I need to make any changes! Thanks heaps and heaps :D

1.   Deparaffinise sections in xylene, 2x 5 minutes.
•   Hydrate with 100% ethanol, 2x 10 dips.
•   Hydrate with 70% ethanol, 10 dips.
•   Rinse in distilled water.

2.   Antigen retrieval – immerses slides in EDTA buffer pH 8.5 (1mM EDTA, 0.05% Tween 20) and microwave sections for 20 minutes.

3.   Rinse sections in TBS for 2x 2 minutes.

4.   Blocking steps:
•   Peroxidase – 10 minute 3% H2O2/PBS, rinse with TBS for 2x 2 minutes.
•   Biotin – 10 minute biotin and avidin blocking solution, rinse with TBS for 2x 2 minutes.

5.   Serum blocking: incubate sections with 20% donkey serum for 30 minutes.

6.   Gently blot off the serum and incubate sections with primary antibody overnight at 4C.

7.   Primary Antibodies:
•   Rat anti-mouse FoxP3 - 1:200
•   Rabbit anti-mouse CCR6
•   Goat anti-mouse MIP-3α
•   Rat IgG 2a – 1:1000
•   Rabbit IgG
•   Goat IgG

8.   Let slides warm up to room temperature for 20 minutes. Rinse sections with TBS 2x 2 minutes.

9.   Secondary Antibodies: incubate the following for 1 hour at room temperature.
•   FoxP3: Alexa Fluor® 488 donkey anti-rat IgG
•   CCR6: Alexa Fluor® 555 donkey anti-rabbit IgG
•   MIP-3α: Alexa Fluor® 647 donkey anti-goat IgG

10.   Rinse sections with TBS 2x 2 minutes.

11.   Mount slides with DAPI+ mounting medium.

please check my protocol for me!
« on: November 07, 2008, 12:47:11 AM »

Offline gula

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Re: please check my protocol for me!
« Reply #1 on: November 07, 2008, 11:27:58 AM »
I don't see why you need a peroxidase and biotin block in this system?
gula

Offline richard03

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Re: please check my protocol for me!
« Reply #2 on: November 07, 2008, 08:02:26 PM »
gula is right!

Block peroxidase ONLY if you used HRP based detection system.

Block Biotin ONLY if you used biotinylated antibody or avidin-biotin detection system.

Since you are using immunofluorescent approach, there is no need to block peroxidase nor biotin.

Offline ASluo

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Re: please check my protocol for me!
« Reply #3 on: November 08, 2008, 04:54:47 AM »
Oppss...yes I agree! I must have forgotten to take it out. Thanks heaps for check for me.  ;D

Offline ASluo

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Re: please check my protocol for me!
« Reply #4 on: November 08, 2008, 05:00:54 AM »
I would like to do double labeling on the same tissue section but not sure about the order of steps like when to apply the second primary and secondary antibodies and whether to block with normal serum between the steps. Can you guys help?
I want to label FoxP3/CCR6 (FoxP3: Alexa Fluor® 488 donkey anti-rat IgG/CCR6: Alexa Fluor® 555 donkey anti-rabbit IgG) and FoxP3/MIP3a (FoxP3: Alexa Fluor® 488 donkey anti-rat IgG/MIP-3α: Alexa Fluor® 647 donkey anti-goat IgG).

Many thanks
Annie ;)

Offline richard03

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Re: please check my protocol for me!
« Reply #5 on: November 08, 2008, 08:36:38 AM »
If both primary antibodies are from different species, you can use parallel approach and refer to this protocol.

http://www.ihcworld.com/_protocols/general_IHC/immunofl_double_parallel.htm

Offline ASluo

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Re: please check my protocol for me!
« Reply #6 on: November 08, 2008, 11:01:52 PM »
Thanks Richard so much for the protocol.

Yes, FoxP3 is rat anti-mouse, CCR6 is rabbit anti-mouse and MIP3a is goat anti-mouse. But my secondaries are all donkey anti-something. Still able to use the parallel or mix them together?

Annie :)

Offline richard03

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Re: please check my protocol for me!
« Reply #7 on: November 09, 2008, 03:59:40 PM »
Yes. You can use the parallel approach and mix the antibodies together.

Thanks Richard so much for the protocol.

Yes, FoxP3 is rat anti-mouse, CCR6 is rabbit anti-mouse and MIP3a is goat anti-mouse. But my secondaries are all donkey anti-something. Still able to use the parallel or mix them together?

Annie :)

Offline ASluo

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Re: please check my protocol for me!
« Reply #8 on: November 09, 2008, 08:15:41 PM »
Many thanks ;D

Re: please check my protocol for me!
« Reply #8 on: November 09, 2008, 08:15:41 PM »