I will work with paraffin tissue sections on the testicular tissue and for detection of apoptosis I will use TUNEL fluorescein kit (Biotium brand).
1) Do you recommend Biotium TUNEL kit?
2) The kit is not contain the Proteinase K for permeabilization and I want to use an physical alternative method. What can I use for the best result (citrate buffer etc.) and how can I apply it?
3) Kit's labeling protocol is telling before the mounting samples wash the PBS containing TritonX-100 and BSA (bovine serum albumin). Can I use the human serum albumin instead of BSA?
4) What material should I use for the cover the tissues/slides after the staining procedures (entellan or something) and what type of the slide should I use? Do I need a special slide and coverslip?
5) Finally you can give me some special tricks for this method.
Thank you for your help already.
Here is the kit's protocol:
1. Materials Required but not Provided
• Phosphate buffered saline pH 7.4 (PBS)
• 4% formaldehyde/PBS • Triton™ X-100
• Bovine serum albumin (BSA) or normal goat or bovine serum
• 70% ethanol (optional)
• Deparaffinization solvents (paraffin sections only)
• Proteinase K (paraffin sections only)
2. Preparation of paraffin tissue sections
a) Optional: include an extra sample to perform negative control (no TdT enzyme) TUNEL labeling.
b) Deparaffinize and rehydrate sections according to standard protocols.
c) Wash twice in PBS.
d) Permeabilize sections with 20 mg/mL proteinase K in PBS for 30 minutes at room 37o C. Proteinase K incubation time and temperature may require optimization depending on tissue type. Alternatively, microwave antigen retrieval protocols may be used at this step.
e) Rinse in PBS. Wash 2 x 5 minutes in PBS.
3. TUNEL reaction
3.1 Incubate samples with 100 uL TUNEL Equilibration Buffer (Component 99965) for 5 minutes. a) For adherent cells or tissue sections, cover sample with a Parafilm coverslip to spread buffer evenly over the cells or tissue section.
3.2 Immediately before use, prepare TUNEL reaction mix by adding 1 uL of TdT Enzyme (Component 99964) to 50 mL of TUNEL Reaction Buffer (Component A) for each labeling reaction.
3.3 Remove Equilibration Buffer and add 50 mL of TUNEL reaction mix to each sample.
a) For adherent cells or tissue sections, cover sample with a Parafilm coverslip to spread buffer evenly over cells or tissue section.
b) For negative control samples without TdT enzyme, add TUNEL reaction buffer without TdT Enzyme.
3.4 For cell staining, incubate for 60 minutes at 37ºC, protected from light. Tissue staining may require 2 hours incubation at 37o C.
a) For adherent cells or tissue sections, perform incubation in a humid chamber.
3.5 Wash samples 3 x 5 minutes in PBS containing 0.1% Triton X-100 and 5 mg/ mL BSA (alternatively, 2% normal serum can be used in place of BSA).
3.6 Counterstain samples if desired. Mount samples in antifade mouting medium for microscopy, or analyze cells in suspension flow cytometry.
Nur,
Here are my answers to your questions:
1) I have personally not used this kit. I recommend the Roche In situ Cell Death Kit TMR Red
https://www.sigmaaldrich.com/catalog/product/roche/12156792910?lang=en®ion=US
2) The Roche kit I use can be used with Citrate or Tris Antigen Retrieval, as well as with proteinase K. However, I would recommend using either Citrate or Tris as Proteinase K use generally require more optimization to determine the right concentration and incubation period. If you don't have an antigen retrieval machine, you can simply place your slides in the buffer and put them in a cheap food steamer for 20-25 minutes and then allow them to cool to room temperature for 20 minutes and wash them with 1x PBS.
3) I would not use human serum here. I would stick with BSA, milk or sera.
4) What material should I use for the cover the tissues/slides after the staining procedures (entellan or something) and what type of the slide should I use? Do I need a special slide and coverslip?
I personally like to use Superfrost Plus charged slides with glass coverslips and use Vectastain Mounting Medium with DAPI (I prefer the hardset as it cures and doesn't require anything else to keep the coverslip on the slide)
5) The best tip I can give you here is that the fixation and processing of your tissue is the key. Overfixed tissue will NEVER stain or stain improperly. Under- or unfixed tissue may stain or may not but will look terrible and morphology will be difficult to determine.
Hope this helps!
Renee
Renee R Donahue thank you so much! Your tips will be great for my tests and I am caring about your experiences.
I will ask you a final question. Can I use a standart citrate or tris buffer or else is there a special ratio for that buffers?
For example a standart Citrate buffer (6.2 pH 3.0) is like;
Or for example a standart Tris buffer (1 M, pH 7.2) is like;
Nur,
You can use standard buffers but you want to make sure that Tris buffer (usually Tris/EDTA buffer) is pH of 9.0 and citrate should be equal to 6.0. You want them to be decently fresh or the pH can be altered over time. I personally use the antigen unmasking solutions from Vector H-3301(Tris) and H-3300 (Citrate). They come in 100x solution (use at 1x) and are more stable over time so they can be used for a very, very long time. Wanted to mention also that if you don't have much experience with mounting and coverslipping tissue, I would use the regular version of Vectastain with DAPI (H-1200) instead of the hard set. It is more forgiving for beginners as far as leaving air bubbles over your samples. Just use it first and then seal the four corners of your coverslip with nail polish and let dry, otherwise the coverslip will slide right off.
Good luck!
Renee
Dear Renee,
I will use antifade mounting medium with DAPI for the mounting and be careful about air bubbles.
Yes, I am beginner for this method and I can't thank you enough for your help.
I wish you continued success!
Nur
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