Senescence Detection Kit (ab65351)

I can confirm frozen tissue sections can be used with ab65351Senescence Detection Kit. The kit has been used for frozen skin sections successfully. You can either fix the frozen tissue in the supplied fixation solution or use your preferred fixation of choice. After creating the hydrophobic barrier around your tissue sections, place enough of the staining solution mix to cover your tissue section. You can modify the specific amount of staining solution that you prepare based on how many slides you are staining and how much volume of staining solution you use per slide.

We do not think that the beta-gal activity will survive paraffin processing and do not recommend using the kit for paraffin embedded tissue.
Briefly, the tissue was frozen in liquid nitrogen, and mounted in OCT. The thin sections (4 um) were cut, mounted onto glass slides, fixed in 1% formalin in PBS for 1 min at room temp., washed in PBS, immersed overnight in beta-Gal staining solution. Then you can view under bright field at 100-200X.The staining results from testing can be found in the article below (The reference is also a principal reference describing the senescence marker) Dimri, G.P., et al. (1995) PNAS 92:9363-9367.

I am sorry we have not tested plastic embedded tissue sections with this kit so we may not be able to provide you required details. You can however test this kit by keeping frozen sections as positive control.

Laminin should not interfere with the staining. The stain will bind only to SA-β-Gal. Also, you will have a normal control cell sample, which you would also grow in laminin and perform the same staining. Any blue color development observed in these wells will be the background (and will account for any non-specific laminin staining).

ab65351 (Senescence Detection Kit) wurde nicht mit Suspensionszellen getestet oder dafür optimiert.

Da dieses Kit allerdings benutzt wird um fixierte Zellen anzufärben, könnte das folgende Protokoll vielleicht benutzt werden mit Suspensionszellen:

1) Take out cells from flask, centrifuge them at 1,000rpm for 10min, at 4°C.

2) Discard the supernatant.

3) Wash cells 2 times with 10ml of cold 1×PBS by centrifuging them at 1,000rpm for 10min, at 4°C.

4) Before 2nd wash, count cell number.

5) Suspend cells in cold 1×PBS to a final cell concentration of approximately 1×10 6 cells per ml.

6) Spit 15μl on slides per circle.

7) Leave the slides to air dry in the laminar flow hood (about 90min or more).

8) Fix the slides in ice-cold acetone for 10min in jars (longer time or less time in acetone will affect the quality of the slides and IFA).

9) Drain the acetone and wash thoroughly the slides with PBS----put PBS into Jar, shake for 5min, then discard PBS, repeat 3 times.

10) Dry by blow dryer or leave the slides to air dry, keep the slides in box at -20°C.

Then we would suggest to start in Protocol booklet :

4. Assay Protocol

step:

3. Staining Solution Mix: While the cells are in the Fixative

Solution, prepare the Staining Solution Mix using a polypropylene

plastic tube only. Prepare enough solution for the number of wells to

be stained.

For each well, prepare:

Staining Solution 470 μl

Staining Supplement 5 μl

20 mg/ml X-gal in DMF 25 μl

Wash the cells twice with 1 ml of 1X PBS.

Dieses Protokoll ist nicht von uns getestet und unterliegt deshalb auch nicht der Abpromise Garantie.

Thank you for contacting us.



Our development scientist has optimized this kit to work best at the conditions mentioned in our datasheet. But sometimes, you have to treat expts case by case and it is possible that in your case, the background might reduce with a higher pH. nice day.

Thank you for your email. I am sorry to hear that you are experiencing problems with lot GR79964-1.

We recommend warming the staining supplement as it is in 100X concentration and frozen. It is not unusual this to be observed jelly like due to high concentration. Please warm it and use as recommended on the datasheet.

We have sold many units of this lot and haven't received any complaint. Could you please provide more details about the problem by answering the following questions;

- What passage number was used for cell line?
- Could you provide, the lot number which was working? Did you use same cell samples as with GR79964-1?
- Did you use tissue sections or cells as sample?
- Could you tell us at what temperature the X-gal was stored?
- Could you provide the exact step followed (protocol) when doing the assay?
- Was the experiment repeated?

Thank you very much for your cooperation. I will look forward to hearing form you soon.

Thank you for contacting us. Although we have never tried dehydrating our sections in order to mount them, I think it would be ok to do so, once you have confirmed the blue colour with the X-Gal staining

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Thank you for your enquiry.

I can confirm frozen tissue sections can be used with ab65351Senescence Detection Kit. The kit has been used for skin sections successfully.

Briefly, the tissue was frozen in liquid nitrogen, and mounted in OCT. The thin sections (4 um) were cut, mounted onto glass slides, fixed in 1% formalin in PBS for 1 min at room temp., washed in PBS, immersed overnight in beta-Gal staining solution. Then you can view under bright field at 100-200X.

The staining results from testingcan be found in the article below (The reference is also a principal reference describing the senescence marker) Dimri, G.P., et al. (1995) PNAS 92:9363-9367.

So in essence, you can follow the protocolfrom thedatasheet for frozen sections.

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

Thank you for contacting us.

The source of the kitis not aware of anyone using this kit with another staining. But it should be theoretically possible to do it.My colleagues suggest to do the X-gal staining first, because the other staining might interfere with X-Gal and if you do not see any staining at all, you would not know if there is senescence or not.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.