lung cryosections preservation for immunofluoresence - (Feb/23/2011 )
Hi guys,
Has anyone tried doing immunofluoresence staining for mouse lung? I plan to do alot of staining with lung cryosections but I have a major problem, which is it is frequently difficult to preserve the lung structure.
Here's my protocol:
1. Harvest lung and freeze in OCT using dry ice
2. cut 10um sections
3. store in -80 (my sections are lying in -80 for 3 months already- how long can they last?)
4. take out and airdry for 5 mins
5. fix in acetone (at -20) for 10 mins
6. block with 3% BSA and 0.2% tween-100 in PBS for 30mins
7. add primary abs and incubate in moist chamber O/N at 4 degrees
8. rinse 3x with PBS
9. add secondary abs for 1 hour
10. rinse 3x in PBS and mount with anti-fade
After all the procedures my sections look fine, they are still firmly stuck to the poly-lysine slides. The problem I am facing right now is that whenever I view the sections under microscope, I found that the cell membrane may have broke and there are stringy/ cell debris mess all over. The alveolar architecture is also disrupted.
Do you guys have any tricks of preserving these fragile cryosections? I have found an alternative protocol online which states to airdry the sections for 1 hour. and to dry it again after fixing. I suppose dehydrating may help to preserve structure but I am afraid that there will be adverse effects on antigen recognition by abs.
Any suggestions?
ctye0712 on Wed Feb 23 10:31:50 2011 said:
Hi guys,
Has anyone tried doing immunofluoresence staining for mouse lung? I plan to do alot of staining with lung cryosections but I have a major problem, which is it is frequently difficult to preserve the lung structure.
Here's my protocol:
1. Harvest lung and freeze in OCT using dry ice
2. cut 10um sections
3. store in -80 (my sections are lying in -80 for 3 months already- how long can they last?)
4. take out and airdry for 5 mins
5. fix in acetone (at -20) for 10 mins
6. block with 3% BSA and 0.2% tween-100 in PBS for 30mins
7. add primary abs and incubate in moist chamber O/N at 4 degrees
8. rinse 3x with PBS
9. add secondary abs for 1 hour
10. rinse 3x in PBS and mount with anti-fade
After all the procedures my sections look fine, they are still firmly stuck to the poly-lysine slides. The problem I am facing right now is that whenever I view the sections under microscope, I found that the cell membrane may have broke and there are stringy/ cell debris mess all over. The alveolar architecture is also disrupted.
Do you guys have any tricks of preserving these fragile cryosections? I have found an alternative protocol online which states to airdry the sections for 1 hour. and to dry it again after fixing. I suppose dehydrating may help to preserve structure but I am afraid that there will be adverse effects on antigen recognition by abs.
Any suggestions?
Frozen sections often give problems with tissue morphology. It might be better if you use paraffin embedded samples - they can be more work but would probably give you much better results, and they can be stored for AGES at room temperature.
If you're not confident about preparing your own embedded samples (and provided they're not particular samples such as examples of a disease state), they can be bought relatively cheaply on-line in the form of tissue arrays: US Biomax Tissue Arrays
Then all you need to do is de-wax, probe etc.
Hope this helps!
I haven't worked with lung sections, but there's some things you could try. Like, I would recommend to fix the lung tissue (in 4%PFA at 4C O/N) before freezing, then wash and dehydrate using 20% sucrose at 4C O/N. Then freeze and cut. Air-dry your sections after cut doesn't inhibit antigen recognition, I usually do it for 1-3hrs at RT.
You may also want to increase sample thickness to 14-20um, structures are much better retained. Parafin sections are one option, the question is of course if your antibody recognizes the antigen, wich is more unlikely than in frozen sections.