×
Please leave your comments or questions
 
Fat Body Staining for Gut
Authors: Catherine Lian and Brian Hodge, updated date: , view: 21, Q&A: 0
Tags: Fly and Drosophila

Procedure

  1. Fat bodies are dissected in Schneider’s drosophila medium (1×);

  2. Fixed in 4% paraformaldehyde (PFA) for 45 min at room temperature (ask Mark).

  3. Samples are rinsed, washed with 0.1% Triton X-100 in 1× PBS (1× PBT) (3 times, 10 min/time).

  4. Blocked in 5% horse serum in 1× PBT at 4°C overnight.

  5. Incubate primary antibodies (Rat-anti-TIM, 1:500; Guinea pig-anti-PER, 1:500) at 4°C overnight.

  6. Washed with 1× PBT for 1 hour.

  7. Secondary antibodies (Rat-Alexa 488, 1:500; Guinea pig-Alexa 555, 1:500) were incubated at 4°C overnight.

  8. Washed with 1× PBT for 1 hour.

  9. Stain for 15 min with DAPI (Sigma; 0.1 μg/ml)

  10. Remove DAPI and add PBT.

  11. The samples were mounted in mounting medium mowiol (ask Mark).

  12. All images were captured by a Zeiss LSM780 inverted confocal microscope and were processed in Adobe Photoshop and Illustrator.

Comment
Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.