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Protocol

Immunofluorescence(IF)

Immunofluorescence Solutions and Reagents

A.  MOWIOL (anti-fade agent)  Calbiochem # 475904
1.    Place 6 grams glycerol in a 50 mL tube containing a small stirring bar.
2.    Add 2.4 grams Mowiol and stir to mix.
3.    While stirring add 6 mL ultrapure water and leave 2 hours at room temp.
4.    Add 12 mL 0.2 M Tris, pH 8.5 and 230 mL 1 %Thimerosal (w/v in water).
5.    Incubate in hot water (50 °C) for 10 min. with frequent stirring.
6.    Centrifuge at 5000 g for 15 min. to clarify stirring to dissolve the Mowiol. This can be repeated over
       several hours to get most of the Mowiol into solution. Store as 2 mL aliquots in glass tubes at -20 °C.
       These are stable for about 1 year.
 
 Warm tube to room temperature before use. After use, store at 4 °C for about 1 month. Discard once crystalline deposits are seen in slides or tubes.

Immunofluorescence Protocol


1.    Plate adequate number of cells into 8-well Lab-Tek. II - Chamber Slide. System least 24 hours before
       fixation.
2.    Aspirate medium from wells.
3.    Wash with 1XPBS pH7.4 for two times.
4.    Fix the cells by adding 250ul pre-cold methanol in each well in -20 °C and incubate for 20 min at -20 °C.
5.    Wash the wells once with 0.5 mL PBS, then aspirate. Add a second 0.5 mL PBS to each well and let
        incubate 5 min.
6.    Add 200ul 1% BSA in PBS to each well and incubate 1.5 hours at room temperature.
7.    Wash the cells twice with PBS and aspirate medium.
8.    Add 150ul primary Ab (in PBS + 1% BSA) to each well and incubate for 2 hours at 37°C or 4 °C
       overnight.
9.    Wash the cells with 0.5 mL PBS with gentle shaking for 5 min. at room temperature (do this 3 times).
 
The Following steps should be performed with as little exposure to light as possible

10.    ncubate the cells with 200ul appropriate secondary Ab conjugated to fluorophore Alexa594 or Alexa
         488 from Invitrogen (in PBS + 1% BSA) and incubate for 20 min at room temperature.
11.    Dyes such as Hoechst (diluted from stock to stain the nucleus  (10 mg/mL Hoechst stock solution) of
         this incubation to make the final concentration 1.5ug/ml.
12.    Wash the cells 3 times with 0.5 mL PBS with gentle shaking for 5 min.
13.    Aspirate as much PBS out of the wells as possible.
14.    Place a drop of mounting medium (Mowiol solution brought to room temperature) on to the middle of the
         Chamber Slide. System.
15.    Put appropriate glass coverslips on the top the plate.
16.    Let slides dry at room temperature (overnight in dark) and examine the cells under a fluorescence
         microscope.
 

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