I am experiencing an issue with mouse brain tissue shattering during staining. The mouse brain tissue is fixed with 4% PFA and sectioned to a thickness of 40um using a Vibratome. After sectioning, the samples are stored in a cryobuffer (40% PBS, 30% Ethylene glycol, 30% Glycerol) at -20°C as a cryoprotectant. This problem has not occurred in the past three years, but recently, it persists consistently in all samples.
Even with newly prepared mouse brain tissue, the shattering issue also occurs. The staining process follows a free-floating immunohistochemistry method on a cell culture plate. The protocol involves TBS wash, 0.2% Triton X 100 treatment for 20 minutes, TBS wash, blocking (1% BSA, 5% donkey serum, 5% goat serum in TBS), overnight incubation with primary antibodies in blocking solution at 4°C, TBS or TBST wash, 2-hour incubation with fluorescent conjugate secondary antibodies at room temperature, TBS or TBST wash, TrueBlack sol (Lipofuscin Autofluorescence Quencher) treatment, TBS wash, and mounting.
While the protocol may vary depending on the target or kit used, the mentioned steps are fundamental. Previously, I did not encounter such issues with tissue shattering, and the staining process went smoothly. The tissue shattering problem only becomes apparent the day after staining initiation or two days later.
In my efforts to resolve this issue, I have tried the following troubleshooting steps:
1. Ensuring all buffers are freshly prepared and using both TBS-based and PBS-based buffers.
2. Preparing new mouse brain tissue and sectioning using 4% PFA, 30% sucrose, OCT, and cryostat methods.
3. Adding an additional 10-minute fixation step with 4% PFA before the staining process.
4. Using a fresh blocking buffer and comparing it with commercial blocking buffers (e.g., Thermo SuperBlock, IHC-TEK).
5. Comparing different primary antibodies with antibody-free blocking buffer and using Fluorescent conjugated primary antibodies.
6. Having a different user perform each step.
Unfortunately, none of the troubleshooting steps mentioned above have been successful. Each condition was tested using different sets of mouse brain tissue (at least two samples for each condition), and control groups were established. Tissues were transfered by using a paintbrush from the cryobuffer to in buffer of cell culture plate, and I ensured gentle handling to avoid any physical damage during the washing or buffer exchange steps.
The problem seams to be the process of sectioning. Play with the combinaton of speed and amplitude of the vibratome. Maybe the speed is to fast and/or the amplitude to low or the other way round. The razor blades you use for sectioning could be the reason. If you have recieved blades from another company you have to check the correct angle of the knifes. I recommand to run some test series to find out what is the best. And before you start with an immuno-test use a simple staining like cresyl violett. This staining is easy to be done an will helps you to find out whether your problem is solved. Good luck!
Ute Neubacher
Hi Ute, thank you for your suggestion. However, I don't think that this is not sectioning problem. There is no problem three months ago.
The question is: Has anything changed with your vibratome since these last 3 months?
Hello Yi Sak Kim,
another reason for your tissues problems could be unsufficient fixation. Maybe the tissue is to soft. you can try to solve the problem with those factors which I have mentionend above. Did you perfuse your animals? If yes, leave them for 24-48 h in the fixation solution before you start with your cryoprotction. And you can add 0,25% glutaraldehyde to your 4%PFA- solution to get the tissue firmer.
Ute Neubacher
Hello Ute,
What I think is different from 3 months ago is that I used freshly made TBS, 0.2% Triton X-100, and blocking solution. However, I tested all new buffers again and the problem still persists.
Yes, my sample was perfused, and sufficiently fixed for more than 48 hours. That's why I don't think I've had any of these problems before.
Thank you for your suggestion. Do you have any other idea?
Now, I'm thinking about microbial contamination.
The parts of my pipettes were autoclaved and treated with UV and 70% EtOH for sterilization.
I have used 1X TBS buffer (not autoclaved) diluted from 20X commercial TBS buffer, and I'm planning to do using autoclaved 1X TBS buffer containing 0.05% sodium azide because I don't need to use HRP-detected methods.
Do you think that is it worth to do test?
Dear Yi,
You need to compare carefully the previous procedure with no problems and the current one with tissue shattering. Some kind of chemical reaction occurs during staining which fragments your tissue and it should be the reason why it happens consistently to all specimens. You did excellen trouble shooting but if not successful, you were unable to eliminate the cause of the reaction. Since it occurs one or two days after you start, there are two possibilities: (a) the process starts at an early step but it takes time to see the effect with catastrophic outcome; (b) it starts at a later step and the effect occurs fast.
You should examine your sections carefully during time to catch eventual early signs of fragmentation to identify the phase of staining which is responsible of the phenomenon. A research study into the study. Histology is always surprising even after decades of experience.
it would be interesting to know when you will solve the enigma.
Best,
Giorgio
Hello Yi Sak Kim,
The problem may rely on before staining or even sectioning. Previously, I had a same problem. I have noticed that some mistakes on transcardiac perfusion, post fixation and tissue storage conditions. The perfusion rate, fixation time, sucrose solution rate and microbial or fungal contaminations... All of them may be a risk factor. Maybe you should control these steps for 3 months before and now.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes