Protocols

Attention
Please test all of the following protocols on a single expendable sample first!

Protocols are not universally valid - they have to be (slightly) adjusted to match different cell types and the protein(s) of interest that shall be stained.
Adjust carefully the concentrations of your antibodies (check the according manuals).
Otherwise you may end up with a large waste of time, material and even loss of irrecoverable samples.
Select you protocols here:

Antigen retrieval in citrate buffer to unmask an epitope altered by fixation

Materials:
  • 10 mM Na-citrate buffer (in most cases pH=6.0 is used, but pH= 9.0 or even 12.0 may also be working, dependent of sample and/or epitope) 
  • Water bath (temperature controlled)
  • Staining chamber (a box containing your samples on object slides)
Procedure:
  1. Heat water bath to 95 °C (203 °F) 
  2. Fill the Na-citrate buffer in the staining box, heat it up to 95 °C in the water bath
  3. Put your samples (deparaffinized, on object slides) into the staining box containing the hot Na-citrate buffer
  4. Incubate for 10-40 minutes
  5. Take the staining box out of the water bath, cool it with tap water to about 50 °C (or less)
  6. Attention: Taking all object slides at the same time out of the hot buffer may cause drying of your samples! This may damage the samples and/or result unspecific (auto)fluorescence.
  7. Wash your samples one by one carefully in aquadest for 3 minutes, each.
  8. Now block nonspecific antigens and immunostain your samples.

Protocol according to “Romeis - Mikroskopische Technik” (German), 19th edition, ISBN-13: 978-3642551895.


Indirect immunostaining of adherent cells

Materials:
  • Phosphate buffered saline (PBS), pH=7.4; weigh 8.0g NaCl, 0.2g KCl, EITHER 1.42g Na2HPO4 OR 1.78g Na2HPO4*2H2O, 0.27g KH2PO4 and fill up to a volume of 1 liter with aquadest; pH should be at 7.4 
  • Permeabilizing solution: 0.2% (v/v) Triton X-100 in PBS
  • Fixing solution: 2% (w/v) formaldehyde (or paraformaldehyde) in PBS
  • Blocking solution: PBS containing 5% (v/v) normal goat serum and 2% (w/v) bovine serum albumin (BSA)
  • Embedding solution: 90% (v/v) glycerine in PBS (for permanent samples you better use an antifade mounting medium, available with and without DAPI)
  •  Ice
Procedure:
  1. Culture your cells on cover slides 
  2. Fix the cells with fixing solution for 5 minutes at room temperature
  3. Wash the cells three times for 5 minutes each with PBS
  4. Permeabilize the cells with permeabilizing solution for 5-10 minutes (on ice)
  5. Block nonspecific antigens with blocking solution for 1h at room temperature
  6. Incubate cells with the primary antibody (diluted in blocking solution at a concentration according to the product specifications) for 1h at room temperature
  7. Wash the cells three times for 5 minutes each with blocking solution
  8. Incubate cells with the fluorescence labelled secondary antibody (diluted in blocking solution at a concentration according to the product specifications) for 1h at room temperature
  9. Wash the cells three times for 5 minutes each with PBS
  10. Embed with embedding solution (or mounting medium)

The steps 2-4 may be also replaced by fixing in cold acetone (-20 °C) or in methanol (-20 °C) for 10-20 minutes each. My personal favorite is a 1:1 (v/v) mixture of ethanol and dimethyl ether (-20 °C, NOT more than 3-5 minutes!). 

If there are problems with staining of membranbe-bound proteins, skip permeabilization and/or don't use lipophilic agents (like ether) for fixation of the cells.

Protocol according to “Romeis - Mikroskopische Technik” (German), 19th edition, ISBN-13: 978-3642551895.

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