Dear Erman Salih Istifli,

Pepsin is the very important component because it's a protease and acts to digest proteins into peptides and it helps to digest away cytoplasm on the slide. Digestion with pepsin can significantly improve probe penetration. Pepsin treatment is often useful when specimen preparations are suboptimal,e.g., in many clinical samples.

Best regards,

Maxim

Dear Maxim,

Many thanks for your kind answer. Which type of cells/cell lines do you use in your studies?

Best wishes

Dear Erman,

We have experience with pancentromeric and whole-chromosome FISH in human peripheral blood lymphocytes and human fibroblasts. We treated lymphocytes by 0.01% pepsin solution for 5 min at +37°С. For fibroblasts, 0.01% pepsin solution for 10 min at +37°С were used.

Dear Maxim

Thanks for your answer. I will mix pancentromeric and pantelomeric probes simultaneously. Do you have a protocol for this kind of application?

Best wishes

Erman

Dear Erman,

We used the following protocol:

At the first stage, you need to prepare the following solutions:

1) 50 ml freshly prepared 1% paraformaldehyde solution (0.5 g desiccated paraformaldehyde + 50 ml 1X PBS + 5 µl NaOH) – you mustn’t store it – only fresh prepared before each experiment.

2) 1000 ml 20X SSC (175.3 g NaCl + 107 g/88.2 g/85.9 g Na3C6H5O7 x 5,5H2O/ Na3C6H5O7 x 2H2O/Na3C6H5O7 x 3H2O), pH = 7. As soon as you dissolved all components you need to filter it. You can store this solution at the room temperature for a long time.

3) 100 ml freshly prepared 2X SSC (20 ml 20X SSC + 180 ml H2O) pour into 3 staining jars Schifferdecker Type and put they into heating oven at +37°С.

4) 70 ml 0.01% pepsin solution (70 µl 10% pepsin + 70 µl HCl concentrated + 70 µl H2O) pour into staining jar Schifferdecker Type and put it into heating oven at +37°С.

5) 1X PBS, pour it into staining jar Schifferdecker Type at room temperature.

Then you need to prepare slides, and after that you can start your experiment.

1) Three-times washing of slides in 2X PBS (5 min, +37°С);

2) Treatment by pepsin solution (5 min – for lymphocytes, 10 min – for fibroblasts, +37°С);

3) Fixation in 1% paraformaldehyde solution (10 min, room temperature);

4) Washing in 1X PBS (5 min, room temperature);

5) Drying in 70% (5 min, room temperature), 80% (5 min, room temperature) and 96/100% (5 min, room temperature) ethanol;

6) Air drying of slides (also you can use Slide warmer if you have this equipment – it can help you to safe your time);

7) Applying 10 µl FISH probes under a coverslip and sealing the the coverslip with rubber cement;

8) Hybridization step. Place the slides in a hybridization system (we use the ThermoBrite StatSpin). Program this equipment in the Denat and Hybr mode (5 min at 75°C and next 16-24h at 37°C) in condition of increased humidity;

9) Preparing the fresh WT1 (2 ml 20X SSC + 98 ml H2O + 300 µl NONIDET P-40) and WT2 (10 ml 20X SSC + 90 ml H2O + 100 µl NONIDET P-40) solutions, pour them into staining jars Schifferdecker Type;

10) Put the WT1 (closed using cover) in water bath at +71-72°C and wait the turbidity of the solution;

11) Washing slides in WT1 (in water bath) for 2 min.

12) All following steps must be performed in dark. Washing of slides in WT2 (room temperature) for 5 min;

13) Washing slides in water (room temperature) for 3 min;

14) Drying slides;

15) Applying one drop of Prolong Gold antifade with DAPI/ Vectashield H-1500 with DAPI under a coverslip, removing the air bubbles and sealing the the coverslip with rubber cement. Prolong and Vectashield protect the slides against oxidation and safe the fluorescent ability;

16) Waiting for 5 min.

17) Analyzing slides using fluorescent microscope.  

I hope it'll help you! If you'll have any questions - you can ask it!

Dear Maxim 

Many thanks for the protocol. Can proteinase K be used instead of pepsin?

Best wishes

Erman

Dear Erman,

Yes, it's possible. Actually, we havn't use proteinase K, but generally incubating the slides with up to 500 μg/ml proteinase K, for chromosomes and isolated nuclei - up to 1 μg/ml for 7.5 min (the optimal amount depends on type of cell and tissues and must be determined) in 20 mM Tris-HCl, 2 mM CaCl2, pH 7.4, for 7.5–30 min at 37°C is used. I think, other RG users who used proteinase K can give you more accurate concentration, or you can try to find it experimentally. Also there is information about less efficiency of this reagent compared to pepsin.

Dear Maxim 

Many thanks for your reply. I am going to try with pepsin. I think it is the most suitable one for FISH. Also, I would be glad if you could answer further questions.

Best wishes 

Erman

How about using Trypsin instead of Proteinase K or Pepsin?

Will that work?

Regards

Karim.