XU LAB CRYOSECTION IMMUNOFLUORESCENCE PROTOCOL V2.1

Thaw slides at room temperature

Outline sections using a PAP pen to reduce reagent usage later

Wash 3X 5 min in wash buffer

For boiling citrate antigen retrieval:

For base/glycine/SDS antigen retrieval:

For HCl antigen retrieval:

NOTE: For multiple antigen retrievals, rinse 2X 5 min in wash buffer between each

Wash slides 3X 5 min in wash buffer

Prepare blocking buffer

Block slides for one hour at room temperature (75 uL)

Prepare primary antibody solution

Blot off blocking buffer

Add primary antibody to slides (75 uL)

Incubate at 4˚C overnight

Wash slides 4X 7 min in wash buffer

Prepare secondary antibody solution

Add secondary antibody to slides (100 uL)

Let sit for one hour at room temperature

Wash slides 3X 5 min in wash buffer

Blot off moisture – *don’t let tissue dry out*

Add one drop of mounting solution to slice

Quickly add cover slip, making sure no air bubbles are present

Optional: apply nail polish around edge of cover slip to slow evaporation

Store at 4˚C

IMMUNOFLUORESCENCE BUFFERS/SOLUTIONS

Wash Buffer (1X PBST/TBST 0.2%)

100 mL 10X PBS (or 50 mL 20X TBS)

900 mL deionized water (or 950 mL)

2 mL Tween 20 or Triton-X

Antibody Buffer

200 mL deionized water

2.25 g NaCl

1.5 g Tris base

2.0 g BSA

3.6 g L-Lysine

2 mL 4% Azide in water

pH to 7.4

Blocking Buffer

50% Goat/Donkey Serum

50% Antibody Buffer

Unmasking Solution (Citrate)

2.94 g Sodium Citrate

DI water to 1 L

pH to 6.0

Base Solution (1% NaOH, 1% H2O2)

2 g NaOH

6.6 mL 30% Hydrogen peroxide

QS DI water to 200mL

0.3% Glycine Solution

0.6 g Glycine

20 mL 10X PBS or TBS

QS DI water to 200 mL

Unmasking Solution (0.3% SDS)

6 mL 10% SDS

20 mL 10X PBS or TBS

QS DI water to 200 mL

Unmasking Solution (1:11 HCl)

187 mL DI water (add first)

17 mL 37% HCl

10X PBS (Phosphate Buffered Saline)

Sodium Chloride (NaCl): 80 g

Potassium Chloride (KCl): 2 g

Sodium Phosphate Dibasic, Anhydrous (Na2HPO4): 14.4 g

Sodium Phosphate Monobasic, Monohydrate (NaH2PO4 H2O): 24 g

HCl / NaOH:  QS to pH 7.4

dH2O: QS to 1 L

Total: 1 L

20X TBS (Tris Buffered Saline)

Sodium Chloride (NaCl): 160 g

Tris Base (C4H11NO3): 48.4 g

HCl / NaOH: QS to pH 7.6

dH2O: QS to 1 L

Total: 1 L

(QS = quantity sufficient for final volume/pH)

I would avoid antigen retrieval with frozen sections - not necessary, and as well, can cause the sections to fall off of the slides.

Thanks every one. 

I will try your suggested protocol and will share my experience once finish the staining.

Hello

agree with Liz Unger that good protocol , Also . find links here will help you

Good luck

http://www.pharma.uzh.ch/research/neuromorphology/researchareas/neuromorphology/Protocols/protocole_immuno_rev.pdf

https://ugpathsoc.wordpress.com/2013/11/11/immunohistochemistry-a-key-technique-for-neuropathology/

http://web.qbi.uq.edu.au/microscopy/wp-content/uploads/2011/pdfs/QBI_HISTOLOGY_AND_MICROSCOPY_GUIDE.pdf

http://publish.uwo.ca/~jkiernan/faqlist.htm

Hi Ajai,

I will just add to Liz recommendations two more antigen retrieval options, since, I don;t know the specifics proteins you are aiming for. However, in my lab we always start with the citrate retrieval.

1) Trypsin retrieval: works great for myelin peptides immunostaining:

0.1% trypsin, 0.1% CaCl in 0.05 M Tris pH 7.4, for 10 minutes at 37˚C

2) Image-iT® FX Signal Enhancer from Invitrogen (cat # I36933) 

Its the last option if you are afraid of losing you tissue by exposing it to heat. Incubation is 30min at RT.

Then, for antigen retrieval I prefer a rice cooker than the microwave. Two reasons:  I don't need to get upset if there is some agarose left-over or competing with agarose people. But the main reason I find it more consistent and you can process more slides at once avoiding handling discrepancies. However, you can use regular coplin jars. Check on Ted Pella for staining dish and slide holder cat # 21078 and 21078-1. They tolerate up to 85˚C. Also, they have no metal which is not good with some of the retrieval buffers.

 For humidified chamber, don't wast your money in those expensive ones. You could do just find with a plastic container with lid paper towel soaked in PBS and chopsticks. Or a plastic slide saver box with hinged lid, again paper towels soaked in PBS and laying the slides on top of the grooves. No need for chopsticks this way. 

In may lab we also use another washing buffer when we do fluorescent microscopy that helps to see the red fluorochomes better. It has fish skin gelatin, some people don't like it cause the smell but to be honest red spectrum could not look better. We got the gelatin from Sigma cat # G7765, 0.2% FSG in 1X PBS.

Lastly, you might need to titrate the serum you use for blocking. Liz included in her protocol 50% of either goat or donkey. However, for some iHC we even go down to 3-5% of goat serum and 10% for donkey. 

Good luck!

Lillian

Yes, I agree with Lillian. When I say "humidified chamber," I mean an old slide box with wet paper towels. You can also use a tupperware with serological pipettes taped to the bottom.

I also agree that 50% serum is excessive, and I have used 5% which works just as well.