[Histonet] Paraffin Tissue Crumbles

[Michael Mashore]

Hello Histonet Users,

 

I have just started using paraffin and am having many difficulties. Most of
the time my tissue crumbles when sectioning. I have no real experience in
paraffin histology and have been given the task of becoming proficient by
myself, so I am hoping for feedback as to why my tissue keeps crumbling. The
tissue in question has been:  skeletal muscle, cardiac muscle, liver, and
brain (all from rat).

 

The tissue was fixed in 10% neutral buffered formalin for 7 days at 4°C and
then transferred to an automated tissue processor, with the following
schedule:

 

2 hours 70% dehydration alcohol 

2 hours 80% dehydration alcohol

2 hours 95% dehydration alcohol

2.5 hours 95% dehydration alcohol

2 hours 100% dehydration alcohol

2 hours 100% dehydration alcohol

2 hours 100% dehydration alcohol

.5 hour Hemo-De

.5 hour Hemo-De

.5 hour Hemo-De

1 hour paraffin

4 hours paraffin

 

They were infiltrated for 1 hour without vacuum then embedded.

The blocks were stored in the freezer before cutting.

The knife angle was 5°. 

Sections were 5µm thick.

 

I would appreciate any feedback whatsoever. 

 

Thank you very much.

 

Michael