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Problem with microtome - (Dec/01/2010 )

Hi everyone,

I apologize for double posting, I posted this topic in Histology section, but I figured it might get more views in the general lab techniques section, so here goes...

my lab has been having some issues with our histology protocol. In trying to optimize to save reagents and time, we have so far have been using the following

Step 1: Dehydration

1. Remove sample from formaldehyde and immerse in distilled H2O 30 min
2. Transfer sample to 70% ethanol 30 min
3. Transfer sample to 80% ethanol 30 min
4. Transfer sample to 95% ethanol 30 min
5. Transfer sample to 100% ethanol (Batch 1) 30 min
6. Transfer sample to 100% ethanol (Batch 2) 30 min
7. Transfer sample to Xylene Batch 1 30 min
8. Transfer sample to Xylene Batch 2 30 min
9. Transfer sample to paraffin boat I in the oven (preheated at 65C) 40 min
10. Transfer sample to paraffin boat II 40 min
11. Transfer sample to paraffin boat III 40min

Step 2: Paraffin embedding

1. Paraffin dispenser should be preheated several hours in advance.
2. Hold the specimen with tweezers in desired position at the bottom of the metal tray and pour paraffin over the sample until it is embedded.


Now here is where we run into problems. When slicing the embedded sample in a microtome, the sample is lost from the paraffin slices (due to being too brittle I guess?) Does anyone have any idea how to modify the dehydration protocol to prevent this from happening? Maybe less time in xylene?

This has been an ongoing issue, so any advice would be greatly appreciated!
Thanks!

yuriy

-yuriy-

your protocol seems to be fine with me
but what do you mean by sample is lost??


yuriy on Wed Dec 1 19:23:54 2010 said:


Hi everyone,

I apologize for double posting, I posted this topic in Histology section, but I figured it might get more views in the general lab techniques section, so here goes...

my lab has been having some issues with our histology protocol. In trying to optimize to save reagents and time, we have so far have been using the following

Step 1: Dehydration

1. Remove sample from formaldehyde and immerse in distilled H2O 30 min
2. Transfer sample to 70% ethanol 30 min
3. Transfer sample to 80% ethanol 30 min
4. Transfer sample to 95% ethanol 30 min
5. Transfer sample to 100% ethanol (Batch 1) 30 min
6. Transfer sample to 100% ethanol (Batch 2) 30 min
7. Transfer sample to Xylene Batch 1 30 min
8. Transfer sample to Xylene Batch 2 30 min
9. Transfer sample to paraffin boat I in the oven (preheated at 65°C) 40 min
10. Transfer sample to paraffin boat II 40 min
11. Transfer sample to paraffin boat III 40min

Step 2: Paraffin embedding

1. Paraffin dispenser should be preheated several hours in advance.
2. Hold the specimen with tweezers in desired position at the bottom of the metal tray and pour paraffin over the sample until it is embedded.


Now here is where we run into problems. When slicing the embedded sample in a microtome, the sample is lost from the paraffin slices (due to being too brittle I guess?) Does anyone have any idea how to modify the dehydration protocol to prevent this from happening? Maybe less time in xylene?

This has been an ongoing issue, so any advice would be greatly appreciated!
Thanks!

yuriy

-Rnotk-

Just to clarify, the sample is so brittle that it turns into dust when sliced in a block. It does not appear in section.

-yuriy-

Longer incubation times might help, but if anyone can post an alternative protocol that would be great..

-yuriy-

dont know what you mean by "longer incubation"
but I just assume that when you cut section, the tissue fall a part, (or came off from paraffin)

if this is the situation, longer hydration might help,
so after you trim the paraffin block, put the block into ice-cold water for a while (5-10min depending on the size of the tissue)
then you can try to cut.

if this wont work, now put the block in warm water to hydrate the tissue before cutting

yuriy on Mon Dec 6 15:53:48 2010 said:


Longer incubation times might help, but if anyone can post an alternative protocol that would be great..

-Rnotk-