Protocol I’ve used :

- Place a sacrificed mouse on its back. Pin the legs and spray the ventral side with 70% ethanol.

- Starting from the mid-abdomen, use scissors to cut along the midline, through the ribcage, and up toward the salivary glands. Expose the trachea by removing tissues or muscles surrounding the trachea.

- Place two threads behind the trachea. Tie one, the highest possible (to avoid the reflux of the ink in the mouth and nose of the mouse). Prepare the other thread to be tied lower around the trachea without tightening the knot.

- Rotate the platform 180°. Use a 1 mL syringe to inject a solution of 15% ink (diluted in distilled water) into the lungs via the trachea between the two knots (we used “aquatint” blue instead of India Ink).

- Tie the second thread and use scissors to cut the trachea and remove the lungs out of the mouse. Remove the heart and the thymus.

- Transfer the lungs into a 15mL Falcon, containing 5 mL of Fekete's solution. The tumor tissue will emerge as white nodules on the blue lungs after a few minutes.

- Wash 3-4 times in the Fekete’s solution in order to remove the excess of ink. Place the tube à 4°C overnight.

- Wash intensively with PBS 1x, count the metastasis under a magnifying glass.

Fekete’s solution receipe :

- 40 mL glacial acid acetic

- 80 mL formalin

- 580 mL ethanol 100%

- 200 mL water

Hi Celliam

I think you can try (at the end of your work) to rinse the lungs into acid alcohol (glacial acetic acid and ethanol in a ratio of 1: 10).

Please if you try, give me your feedback.

Thanks and best regards.

Azza Zidan.

Hi Azza,

I'll try next time (in one month), if this is the only solution you think that can help me.

Thank you for your answer.

Célia

I agree with doing a rinse after collecting the lungs. We always transfer the lungs to di water for 5 minutes and gently shake to remove as much excess blood and ink before moving to Fekete's for overnight fixation.

Hi everyone, 

Thank you for your answer Adam and Azza, we finally found why our coloration wasn't lasting. Actually it's because we didn't use India ink but a chemical blue tint. I think that using real India Ink it's the most important part of this lung coloration.

I also try with or without washing the lung, and you're right, washing improve the contrast between the lung and the metastasis.

Thanks anyway for your help.

Célia

thanks you for your help ... i did it in a very nice way ...but i have a question after putting the india ink ... did you wash the lungs with Di-water or (glacial acetic acid and ethanol in a ratio of 1: 10). ???

I do a quick (~5 min) wash by setting in di-water, followed by permanent storage in Fekete’s solution. Célia gave a Fekete's solution recipe above.

Yes it's very important to remove as much excess blood and ink before moving to Fekete's for overnight fixation. 

I was wondering if it is possible to use the India Ink assay to detect pancreatic tumors in the pancreas and not just the lungs?

Hello everyone,

I am about to follow this protocol to visualize lung metastasis (TC1, IV injected).

I have a lot of mice which means a lot of time to collect all the lungs. However, I cannot place the lungs straight into the Fekete's solution, since it is toxic and I cannot use it outside of a chemical hood while I am still at the procedure room. Can I put the lungs in cold PBS or di water until I am done with the collection of all the lungs (~1 hour)? Do you have any suggestion on that?

Thank you very much in advance.

Nektarios, that should be fine. I always place in water first for about 10 min and even do a bit of very gentle agitation and rinsing to remove any blood or ink on the lung surfaces before placing in Fekete's. Otherwise, any blood/ink will cover the lung surface (and mets). I would highly recommend practicing on some mice first. This is not an easy procedure and takes a few times to get really comfortable.

Thank you very much for your advice. I will practice on some mice first before moving forward with the actual experiment. Thank you!

Hi, I have not worked on mouse model yet. I am in the process of writing proposal and getting approval approval for animal work. My question is, if we can perform hematoxylin and eosin staining after we stain the lungs with India ink.

Sushila, yes H&E works perfectly on ink-stained lungs. There may be a very small amount of ink at the tissue edges, but not penetrating the tissue. Immunofluoresence, however, did not work well for us on ink-stained samples.

Thanks a lot Adam. This is really helpful.

15 percent ink solution in PBS or Water ?

Hi Sachin,

I diluted india ink in water but I believe it would work as well if diluted in PBS.