Have you try to decrease the formalin-fixed time?

Have you try to decrease formalin-fixed time?

So many variables here. Cold wax blocks will definitely shatter, and if you are inexperienced in sectioning you might have more success slicing at 8-10 microns. If the tissue itself is brittle in a room-temperature block, examine your tissue prep. Dissect smaller blocks of tissue and/or increase time in all the dehydration or clearing steps. Embed a softer tissue like liver, kidney, gut or testis in the same block with your tendon and see if it breaks or sections well. Also try heating your wax 1-2 °C higher than before to improve infiltration; semisolid wax does not penetrate well.

Thanks so much. I have significant experience sectioning heart, kidney, and aorta. I definitely circulated hearts at the same time as these tendons and had no issues sectioning them. In my experience, I have a much easier time and get better sections heart and aorta when the blocks are cold. Kidney and liver however...need to be room temp - so this is why I tried both methods with the tendon. It is definitely the tendon itself that is brittle, so I would be hesitant to increase dehydration time. My concern is actually that the tissue was dehydrated too much (given that the water content in tendon is already considerably lower than other tissues that I routinely process).

I have occasionally heard that there are different tricks to soften brittle tissues after they have been embedded in paraffin. I just don't know what those tricks are.

As for size of tissue...this is rat achilles...I'm not sure I could get much smaller. There's not a whole lot of tissue to work with. Fixation was overnight.

i have read about using Downy fabric softner for soaking paraffin blocks that are dry, brittle, and hard to cut.i never tried this though .

REAGENT:

Downy Fabric Softener 5.0 ml (obtain through Hospital Stores)

Distilled water 100.0 ml

Mix well

PROCEDURE

1. Face into block.

2. Place face down into solution.

3. Allow to soak for 5-10 minutes.

4. Can be used to wipe the face of the block prior to each section.

a. Squeeze out all excess solution from Kim Wipe.

b. wipe face of block, section, repeat for as many sections as needed on ribbon.

Thanks Mohamed! I'm willing to try it. I have a bunch of blocks that I can experiment with. I also recall someone using Nair on their blocks...but I can't remember what the reason was.

Hi Taben

brittle tissue during cutting means incomplete dehydration, clearing or infiltration. So, try to adjust these processing times again, if you have more samples, especially when processing tissues manually. Also, try to change the cutting angle because it differs slightly from soft tissues and hard tissues like collagen. Also, chilling the face of the paraffin section by ice cube may give you 2 to 3 serial sections 6 to 7 µ each.

Best regards

Thanks all for your input. I'm curious about the processing and potential incomplete dehydration, clearing, or infiltration. I had read on a different Q&A forum that tendon is susceptible to "over dehydration" because it has a relatively low water content. In that forum they had suggested to the poster that they should shorten their processing times. This is my protocol (performed in an automated benchtop processor):

Methanol 50%, 30min,

Methanol 100%, 30 min,

4 X methanol 100%, 45 min,

3x toluene 100%, 45min,

parraffin: 30min, 60C

parraffin: >45 min, 60C

Since this runs overnight, the cassettes typically are in the last paraffin bath for several hours. This is our standard protocol...but I would be open to suggestions on modifying it.

Water and wax and not compatible, so you want total dehydration, but minimal post-dehydration processing. For small samples, we place in 70% ethanol overnight, then next day make 2 changes of 95% EtOH, (30 min each), 2 changes of 100% EtOH (30 min each), 2 changes of xylene (1 hour each) and 2-3 changes of wax (45-60 min each). Try a much shorter time in wax and see if it helps.

Thank you all very much!

When I have trouble with sections that brittle, I first face the block and then place the block in the water bath that I use for expanding the sections before mounting. 10-15 seconds is usually enough. Then I cool the faced/rehydrated block in the freezer (-20C) for about 5 minutes. This is usually enough to get a nice ribbon of sections without too much hassle.

Joan - thanks for the tip. I'll try that. I tried putting the block face down in warm water...and that wasn't successful, but I will try re-cooling after hydrating as you suggest.

Hi Taben.

Would you mind to share with us which tips had worked with the tendons you're working with?

Thanks!

Emilio

Hi Emilio,

I am so sorry for taking so long to respond...I don't sign into Research Gate very often. What ultimately worked best for me was soaking the block face down in Downy fabric softener (5ml Downy in 100ml distilled water).  I got this tip from the attached document.  Best of luck!

Thanks Taben!

Fortunately, this technique also works with elastic marine sponges (Porifera).

Cheers!