I want to image nanoliposome conjugated-GFP in tumor tissue after intratumoral injection by fluorescent microscopy, which freezing method is better after removing of tissue for cryosectioning? Can I transfer the tissue directly to OCT medium or fix it in PFA 4% and sucrose before transfer into the medium? Does fixation by PFA and sucrose reduce the fluorescent effect of GFP? I want to image nanoliposome-GFP accumulated in tumor tissue after removing the tissue, cryosectioning, preparing 10 micrometer section on glass slide and visualize with fluorescent microscopy. which freezing method is better for preparing cryosection so that the fluorescent effect be maintained?
I can highly recommend this protocol, it is annoying but worth it:
Visualization of Fluorescent Proteins in situ (GFP/YFP histo on fixed tissues)
NOTE: Tissues have to be fixed in PLP prior to freezing to prevent diffusion of the highly soluble FP.
1. Preparation of Periodate-Lysine-Paraformaldehyde (PLP) -Fixative
· Prepare 0.1M Na2HPO4 (below “Dibasic”) MW=141.86
Dissolve 14.18 g Na2HPO4 (from JT Baker) in 1000 ml H2O dd
· Prepare 0.1M NaH2PO4 (below “Monobasic”) MW=137.99
Dissolve 13.8 g NaH2PO4 (JT Baker) in 1000 ml H2O dd
· Prepare 0.1M Phosphate Buffer (below “PB”)
Use 3 parts of Dibasic buffer and 1 part of Monobasic buffer.
For 1l of fixative (make fresh! Final concentration: 0.01 M Sodium-M-Periodate, 0.075 M L-Lysine,1% Paraformaldehyde):
·Add 13.95 g L-Lysine (Sigma) to 370 ml H2O dd, adjust pH to 7.4-7.5 with Dibasic.
·Heat 200 ml H2O dd to 60º C. Add 10 g Paraformaldehyde (Sigma) (PFA) while stirring. Clear solution by adding 5N or 10N NaOH (a few drops is enough!). Cool to RT. Bring to 250 ml with H2O dd.
·Add the PFA-solution to the Lysine buffer. Adjust pH to 7.0-7.2 using Dibasic if pH7.2
·Bring the volume to 1000 ml with PB.
· Add 2.13 g Sodium M-Periodate (Sigma).
1. Harvest tissues (protect from light while processing!) and fix overnight at 4º C with gentle shaking (use 50 ml tubes with 20 ml fixative per organ, cut spleens in half).
2. Prepare a gradient of 10%, 20% and 30% Sucrose-solutions in 0.1 M PB (Dissolve 10g, 20g and 30g Sucrose per 100ml PB). Sucrose serves as cryoprotectorant and prevents ice crystals from forming during freezing and thawing, thus preserving morphology.
3. Pour off PLP using a cell strainer and wash tissues 3x for 5 min. with 0.1M PB.
4. Add 10% Sucrose, wait until tissue sinks. Then pour off and add 20%. Let tissue sink again (can take up to an hour), then add 30%.
5. When tissue sinks, remove it and dry surface with Kimwipe. Place OCT in labeled cryomold and embed tissue.
6. Freeze in Isopentane in a Pyrex dish surrounded by dry ice mixed with Ethanol. Wrap in Saran wrap and tin foil and store at –80 º C freezer.
7. Section tissues on cryostat in the dark (this might not be necessary if anti-FP Ab is used later), using positively charged slides.
8. Air dry at RT for 30 minutes.
9. Freeze at –80º C for storage. If samples will be examined right away, rehydrate with PBS for 20 min, then use coverslip with anti-fade mounting medium (e.g. Prolong gold).
10. Weak FP-signal can be enhanced by staining with anti-FP-antibody such as goat anti-GFP-FITC (Rockland # 600-102-215). I tested this Ab for GFP and YFP, working dilution 1:500.
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