Hello everyone,
I am looking to perform histology and immunohistochemistry on rat long bones (mainly femurs and tibiae).
I would like some expert help with my preparation protocol for the sections of bone I am obtaining.
What I do currently is:
- Fix in 10% formalin for 24hrs immediately after extracting the bone.
- Rinse in PBS 10 mins.
- Rinse in distilled water 10 mins.
- Decalcify whole bones in 10% EDTA at 4 celsius at 50ml solution per bone (individual 50ml pots for each bone), constant agitation, change solution 3x weekly, 28 days total.
- Check decalcification end point by ease of bending with fingers.
- Rinse in PBS 30 mins then place in the autoprocessor:
- Dehydration 1.5 hours each in 50%, 70%, 95%, 100%, 100%, 100% (three 100% steps) ethanol.
- Clear in 100% histoclear 2x 1 hour (2 identical steps of 1 hour)
- Wax infiltrate in molten paraffin 2x 3 hours (2 identical steps of 3 hours), no vacuum available.
- Once autoprocessor has finished, wax embed immediately in molten paraffin wax at 70 celsius, allow to set on a cold plate for 20 mins minimum, then slice on a rotary microtome. I have purchased special microtome blades for hard tissue from VWR.
- Slice at 4 microns thickness down to where the depth has reached the marrow cavity (passed through the cortical shell), then soak the exposed face in ice water for 10 mins.
- Slice this softened bone at 4 microns.
- Allow sections to dry at room temp overnight, or in an oven at 50C for 2 hours.
- Then do H&E staining, IHC staining etc.
The main issue I have is that I don't know what part of that process is too long or short. Do I clear for longer? Do I leave in ethanol for shorter? Is my wax too hot? Does oven drying at 50C affect the bones differently to leaving overnight at room temp? Etc. etc.
I want to obtain the best sections possible. Previous attempts have resulted in useless, overly brittle bones that I tried reverse processing and re-decalcifying, but they were un-salvageable and simply wouldn't slice. The current protocol above has achieved nice ribbons of slices, however it is not optimised.
I don't know if its the decalcification that needs fixed, or the processing steps, or a mix of both. I also don't know what I'm checking for when I check decalcification with my fingers (how soft is soft enough?).
I would like to end up with a reliable protocol that gives me the most consistent sections for comparison possible.
My only big thought currently about changing my protocol is to use a bone nibbler to snip each bone in half at its midpoint, forming a proximal and distal end, which I can then align beside each other in the wax cassettes. Cutting in half would decrease the required decalcification time as the EDTA could permeate the inner cavity of each bone much easier. How much would it reduce the decalcification time by? Is it a viable option?
Hi Flynn,
the protocol looks ok to me in general, however there still might be an issue with proper decalcifying or fixation. With these long bones I would give a try to fix them longer, 48hours and transfer them into EDTA directly(washing is not needed in this step) and provide the decalcifying at RT instead of putting them into fridge.
Cheers,
Attila
+ forgot to mention, the working solution for decalcifying is not needed to change, you can use one during the whole procedure.
If the bones are elastic, which basically means you can bend them, they are ready for processing.
You may choose to determine the end-point of decalcification either by (i) trying to cut the tissue with a microtome blade (the less 'crunchy' it is to pass through, the more suitable it is for sectioning), or (ii) mixing used decalcification solution to ammonium hydroxide and oxalate (clear solution indicates decalcification is complete)
(ii)https://www.labce.com/spg942012_chemical_methods_of_end_point_determination.aspx
Hello Flynn,
You may use following protocol to decalcify the bone samples. I worked with rat bone samples ( femurs and tibiae ) and was able to obtain good sections for HE staining.
1. Remove the extra muscle parts using a microtome blade from the bone samples as much as you can when you get the samples for formalin fixation (this will allow the bones to be fixed in formalin well; usually bone samples need at least 2-3 days for proper fixation)
Sample trimming > Ref: https://www.niehs.nih.gov/research/atniehs/labs/assets/docs/q_z/revised_guides_for_organ_sampling_and_trimming_in_rats_and_mice_508.pdf
2. Wash the formalin fixed tissue in running tap water for 30-60 minutes (larger specimens need more washing time as to remove the formalin completely>extra formalin in tissue will react with hydrochloric acid , making tissue harder/brittle)
Prepare solutions as follows:
(Ref: IHC world protocols> http://www.ihcworld.com/_protocols/histology/decalcification.htm)
8% Hydrochloric Acid Stock Solution:
Hydrochloric acid, concentrated ----------------- 40 ml
Distilled water ------------------------------------- 460 ml
Mix well and store at room temperature.
8% Formic Acid Stock Solution:
Formic acid --------------------------------------- 40 ml
Distilled water ------------------------------------ 460 ml
Mix well and store at room temperature.
Hydrochloric Acid/Formic Acid Working Solution:
Combine equal parts of the 8% hydrochloric acid solution and the 8% formic acid solution before use.
Ammonia Solution:
Ammonia, Concentrated ------------------------ 5 drops
Distilled water ----------------------------------- 100 ml
Mix well
3. Keep samples in decalcifying solution (needs solution 20 times their tissue volume> needs optimization to determine the proper time for decalcification>usually femur and tibiae needs overnight incubation in decalcifying solution>in-between trim your samples with microtome blade to check the hardness of the bones, once it properly decalcified ,when you trim it using the microtome blade ,you can feel bones become soft ).
4. Rinse the samples in running tap water for 15-30 min and keep in ammonia solution for 30min.
5. Wash in running tap water overnight.
6. Proceed with embedding
Tips for embedding: Make sure every corner of your tissue samples touch the bottom of the embedding cassette >* important to get equally thick sections
Tips for sectioning: Pre-cut sections using 30um and proceed with 4um serial sections for staining. Take the tissue sections on to glass slides as soon as you see clear surface without folds.
Hope this helps!!
Charith
- Badges
- Science topic
- Similar topics
- Engineering
- Petroleum Engineering
- Gas