Whole Mount Immunolocalization to Fucus




Дата канвертавання19.04.2016
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Whole Mount Immunolocalization to Fucus

Shed Fucus and adhere to microscope slide


Fixation


Fixation solution:80mM PIPES buffer pH 7.4

5mMMagnesium Chloride

20% Glycerol

1mM PMSF


4%Paraformaldehyde
Wash Buffer: 80mM PIPES pH7.4

0.45M NaCl

20% Glycerol
Procedure:

1) Fix tissue for 45 min at RT in Fixation solution

2) Wash 3x5 minutes at RT in wash buffer

3) Change gradually to PBS (5min in each): a) 80% wash buffer + 20% PBS

b) 60% + 40%

c) 40% + 60%

d)20% + 80%

e)0% + 100%

4) 2changes in 100% PBS (5min each)
Cell Wall Digestion:
Solution: mix in 1xPBS

2% Abalone Aceton Powder

2% Cellulase

when “almost” dissolved add 7ug/ml Leupeptine

7ug/ml Pepstatin

1ug/ml Aprotinin

1mM PMSF

Procedure: 1) Select and perform various digestion times (1, 2, 3, 5, 10 min…), also can try 1% Abalone acetone powder and 1% Cellulase instead of 2%

2) Following digestion, remove digest solution and rinse 3x in 1X PBS

3) Incubate 1/2 of each slide in Triton solution (1ml 1X PBS, 7ug/mlLeupeptine, 7ug/ml Pepstatin, 1ug/ml Aprotinin, 1mM PMSF, 0.1% Triton X100

4)Rinse 3x5min in 1X PBS
Immunolocalization:


  1. Apply blocking buffer to each well, keep in humid chamber (wet papertowel on the bottom of Petri Dish ) O/N. Blocking buffer (1ml): 0.01g BSA, 50ul Normal Goat Serum (=1%), 950ul 1XPBS

  2. Remove blocking buffer from O/N slides and apply Primary antibodies in blocking buffer (final conc. 2ug/ml) Apply about 45ul to each well on slide. Incubate at RT for 1 to 2 hours

  3. Remove primary antibodies it a pipette

  4. Rinse 3x 5-10 min each with Blocking Buffer

  5. Remove final wash, add secondary antibodies: Alexa 488 anti rabbit antibodies 1:200 dilution in Blocking Buffer (prepare and keep in dark). Incubate for 1-2 hours at RT in dark

  6. Rinse 3x in Blocking Buffer

  7. Rinse 4x in 1X PBS

  8. Check under microscope. Use mounting solution (Slow Fade, Molecular Probes, Eugene Oregon) and cover slips, Seal with nail polish to prevent desiccation.

References:


Bouget FY, Gerttula S, Quatrano RS (1995) Spatial redistribution of poly(A)+ RNA during polarization of the Fucus zygote is dependent upon microfilaments. Dev Biol 171: 258-261
Bouget F-Y, Gerttula S, Shaw SL, Quatrano RS (1996) Localization of actin mRNA during the establishment of cell polarity and early cell division in Fucus embryos. Plant Cell 8: 189-201


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