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.
 

De-Paraffinization and Antigen Retrieval
1.    Incubate slide at 60°C for 60 min.
2.    Bath the slides in antigen retrieval solution and heat it to boil with a microwave on medium power.
       Once boiling, immediately transfer to a small power and keep faint boiling while heating for 2-3 min.
3.    Cool them to room temperature and wash rocking in distilled water several times.
4.    Immerse slides in 3% H2O2 (in fresh methanol) for 15 min at room temperature.
5.    Wash with TBS (pH 7.6) three times, 5 min each time.
 
Staining with Primary Antibody
1.    Dilute primary antibody with 3% BSA in TBS. Cover the tissue section on the slide with diluted primary
       antibody (use 50 – 150μl for each slide).
2.    Incubate at 37°Cfor 2 hours (The optimal incubation time, incubation temperature, and antibody dilution
       should be determined by the individual laboratory).
3.    Wash with TBS three times, 5 min each time.
 
Staining with Secondary Antibody
1.    Incubate with 100-200μl Polymer Enhancer. Incubate 30 min at room temperature.
2.    Wash with TBS for 3 times, 5 min each time.
3.    Incubate with 100-200μl Polymerized HRP and incubate 30 min at 37°C.
4.    Wash with TBS for 3 times, 5 min each time.
5.    Add DAB solution and incubate 3-5 min(The reaction progress and the optimal time should be determine
       according to microscope).
6.    Wash with distilled water for 2 times, 5 min each time.
7.    Counterstain sections in hematoxylin if required,wash with distilled water.Immerse slides in 0.1% HCl-
       ethanol for 1-10 seconds, wash with distilled water. Doing a counterstain of sections in hematoxylin for
       1-2 min and wash them with distilled water.
8.    Dehydrate through 100% ethanol for 5 min, 90% ethanol for 5 min, 75% ethanol for 5 min, and coverslip
       with mounting medium.

 

Deparaffinization

1) Incubate slide at 60 degree for 60 minutes.
2) Deparaffinize in Xylene for 10 minutes and repeat one more times.
3) Hydrate in 100% alcohol for 5 minutes, in 95% alcohol for 5 minutes, in 85% alcohol for 5 minutes, in 75%
alcohol for 5 minutes.
4) Dip into Distill Water for 5 minutes.
5) Dip into TBS (50 mM Tris, 100 mM NaCl, pH 7.6), leave for 5 minutes, and repeat two times.

Antigen Retrieval

6) Bring 500 - 2000 ml 10 mM citrate buffer (pH6.0) to the boil in a stainless steel pressure cooker.
7) Put the slide into staining rack and lower into pressure cooker ensuring the slide is well immersed in citrate
buffer.
8) When the pressure indicator valve has risen after 3-4 minutes, incubate for 1 minute.
9) Cool the slide naturally to room temperature.
10) Dip into distilled water, leave for 5 minutes, and repeat two times.
11) Dip the slide in TBS for 5 minutes and repeat two times.
12) Immerse slides in 3% H2O2 ( in fresh methanol) for 15 minutes at room temperature.
13) Wash with distilled water two times, 5 minutes each time.
14) Wash with TBS (pH 7.6) two times, 5 minutes each time.

Staining with Primary Antibody

15) Place the slide into Blocking Solution (3% BSA in TBS) for 30 minutes.
16) Dilute primary antibody with 3% BSA in TBS. Cover the tissue section on the slide with diluted primary
antibody (use 50 – 150 μl for each slide).
17) Incubate at 37degree for 30 minutes or at room temperature for 60 minutes (The optimal incubation time,
incubation temperature, and antibody dilution should be determined by the individual laboratory).
18) Wash with TBS two times, 5 minutes each time.

Staining with Secondary Antibody

19) Incubate with 100-200μl biotinylated secondary antibody diluted in Blocking Solution. Incubate 30 minutes at
37degree.
20) Wash with TBS for 3 times, 5 minutes each time.
21) Incubate with 100-200μl streptavidin- HRP and incubate 30 minutes at 37degree.
22) Wash with TBS for 3 times, 5 minutes each time.
23) Add DAB solution and incubate 10 minutes(The reaction progress and the optimal time should be determine
according to microscope);
24) Wash with distilled water for 2 times, 5 minutes each time.
25) Counterstain sections in hematoxylin if required,wash with distilled water.Immerse slides in 0.1% HCl-
ethanol for 1-10 seconds, wash with distilled water .
26) Dehydrate through 95% ethanol for 1 minute, 100% ethanol for 2×3min, Xylene for 2×3min, and coverslip
with mounting medium.

Western Blotting Solutions and Reagents

1.    10X Phosphate Buffered Saline (PBS): To prepare 1 L of 10X PBS buffer: 80g NaCl, 2g KCl, 36.3g
        Na2HPO4•12H2O, 2.4g KH2PO4, adjust pH to 7.4.
2.    1X Cell Lysis Buffer with protease/phosphatase inhibitors
3.    5X SDS Sample Buffer:1M Tris-HCl (pH 6.8), 10% SDS, 50% glycerol, 500mM DTT, 0.05% Bromophenol
       Blue
4.    Transfer Buffer:25 mMTris base, 0.2 M glycine, 20% (or 10%)methanol (pH 8.5)
5.    10X Tris Buffered Saline (TBS):To prepare 1 L of 10X TBS: 24.2 g Tris base, 80 g NaCl; adjust pH to 7.6
       with HCl (use at 1X)
6.    Blocking Buffer:5% nonfat milk, 1X TBS, 0.1% Tween-20
7.    Wash Buffer/TBST:1X TBS, 0.1% Tween-20(100%)
8.    HRP-conjugate secondary antibody
9.    Chemiluminesence reagents

Cell Lysis Protocol

Westerns are performed using cell lysates from harvested cells.
1.    Treat cells by specific regulator for desired time, wash cells with 1X PBS.
2.    Detach adherent cells using a cell scraper or centrifuge suspended cells, and resuspend in 1X Cell 
       Lysis Buffer with inhibitors.
3.    Remove a small volume (50 μl). To perform a protein assay, determine the protein concentration for each
       cell lysate.
4.    To the remaining volume of cell lysate, add half volume of 5X SDS Sample Buffer.
5.    Boil each cell lysate at 100 °C for 5 min.
6.    Centrifuge at 14,000 rpm in a microcentrifuge for 5 min.
7.    Load equal amounts (30–45μg) cell lysate onto SDS-PAGE gels using loading tips, along with molecular
       weight markers.
8.    Run the gel and transfer to nitrocellulose for western immunoblotting.
 
Western Blotting Protocol

Membrane Blocking

1.    Block membrane by incubating 1 hour at room temperature or overnight at 4 °C with shaking in Blocking
       Solution(5% nonfat milk, 1X TBS, 0.1% Tween-20).

Incubation with Primary Antibody

1.    Dilute primary antibody at the appropriate dilution in Blocking Solution.
2.    Incubate the membrane with diluted primary antibody for 2 hours at 37 °C, or overnight at 4 °C with
       agitation.
3.    Remove antibody solution. Wash the membrane 3 times for 5 minutes each time at room temperature in
       TBST with shaking.

Incubation with Secondary Antibody

1.    Incubate membrane with diluted HRP-conjugate secondary antibody (according to manufacturer’s
       instructions) in Blocking Solution for 1 hour at room temperature with shaking.
2.    Remove antibody solution. Wash the membrane 3 times for 5 minutes each time at room temperature in
       TBST with shaking.

Chemiluminescent Reaction

1.    Prepare and use the Chemiluminescent substrate according to the manufacturer’s instructions.
2.    Chemiluminescent substrate lay evenly over the membrane. Put them into chemiluminescent imager for
       exposure.

Peptide Competition Protocol

Before proceeding Western Immunoblotting, add specific Blocking Peptide (Refer to blocking peptide catalog#) to the diluted primary antibody in a molar ratio of 10:1 (peptide to antibody) and incubate the mixture at 4℃ for overnight or at 37 °C for 2 hours.

Phosphatase experiment Protocol

After blocking membrane,wash membrane with TBST for 1-2 min, then add phosphatase to the diluted primary antibodywith the appropriate concentrationand incubate the mixture at 4℃ for overnight or at 37 °C for 2 hours.

 

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