In Situ Cell Death Detection Kit, Fluorescein

TUNEL assay cell type - Islets of langerhans (Beta cells)

Experiment
TUNEL assay cell type - Islets of langerhans (Beta cells)
Product
In Situ Cell Death Detection Kit, Fluorescein from Sigma-Aldrich
Manufacturer
Sigma-Aldrich

Protocol tips

Protocol tips
Fix sample in 10% buffered formalin for 24h before using sample.

Block samples in 10% donkey serum in 0.1% Triton-X for 30 min at room temperature.

Incubate primary antibody overnight at 4 °C and with secondary antibodies for 1 h at room temperature

Publication protocol

At the end of the respective culture durations (24 h, 3 d, 5 d, and 7 d) pelleted islets underwent fixation for 24 h in 10% buffered formalin. The pellet of the fixed isolated islets was then pipetted onto a microscope slide with a circular concave divet in the center. The islets were dispersed throughout the agar as the 2% agar solution was added to the slide. The agar was permitted to harden and then the agar disc was dehydrated and processed to wax through a series of alcohol, xylene, and wax baths. Discs once embedded in paraffin were sectioned at 5 µm thickness.49 Hematoxylin and eosin (H&E) stained histo-processed islets were assessed for physical integrity and cell/tissue turnover (mitosis, necrosis, apoptosis) using the Olympus Compound Microscope by a pathologist who was masked to the study.

Apoptosis was also assessed by the Confocal Microscope LSM 510, Zeiss following in situ hybridization terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) method50 using the In Situ Cell Death Detection Kit (Fluorescein, 1 684 795, Roche Diagnostics Corporation, Roche Applied Science). Pancreatic tumor tissue following therapy was used as the positive control as it has a naturally high apoptotic index. For the negative control the TUNEL reaction mixture contained only label (fluorescein) and for the positive control, the reaction mixture contained label and enzyme.

Endocrine cell composition was assessed by immunohistochemistry (IHC) where the primary antibodies used were: anti-insulin (raised in mouse) and anti-glucagon, anti-pancreatic polypeptide and anti-somatostatin (raised in rabbit), purchased from Zymed Laboratories Inc. The Dako LSAB 2 System Peroxidase (Universal, K0672, K0673, K0675) kit comprised of biotinylated anti-mouse or anti-rabbit with streptavidin conjugated to horseradish peroxidase, developed with 3,3′-diaminobenzidinetetrahydrochloride (Sigma-Aldrich) and counterstained with Harris’ hematoxylin (Sigma-Aldrich).

Full paper   Login or join for free to view the full paper.

Reviews

In Situ Cell Death Detection Kit, Fluorescein from Sigma-Aldrich has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Discussion

5 years ago

Author: Ercole Udinesi Italy

Are 10 week old samples in formalin still usable?

I have left my tumor samples in formalin for an extended period of time (around 10 weeks). Do you think I will be able to use them for TUNEL assay and get results? Thank you for your help in advance

Share your thoughts or question with experts in your field by adding a discussion!

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing TUNEL assay cell type - Islets of langerhans (Beta cells) using In Situ Cell Death Detection Kit, Fluorescein from Sigma-Aldrich.

View full paper   Login or join for free to view the full paper.

Manufacturer protocol

Download the product protocol from Sigma-Aldrich for In Situ Cell Death Detection Kit, Fluorescein below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing TUNEL assay cell type - Islets of langerhans (Beta cells) using In Situ Cell Death Detection Kit, Fluorescein from Sigma-Aldrich. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms