Biology Reference
In-Depth Information
8. The size and microcarrier loading quantity (MLQ) need to be
optimized for each instrument and tissue sample/experimental
preparation. Delivering 0.5 mg/1 ml of gold per target is the
manufacturer-recommended starting point (MLQ = 0.5).
9. The gas pressure should be optimized for each system and tis-
sue sample/experimental preparation utilized. The manufac-
turer-recommended range is 100-600 psi.
10. Eye and ear protection are required as per the manufacturer.
1. Blood and blood products in the tissue contribute to autofl uo-
resce and staining artifact. Thus, in some applications, heparin
(10 U/ml) can be added to the PBS buffer to improve
exsanguination.
2. Dissection tools used to remove PFA-fi xed tissue should never
be used for fresh or live tissue, as residual PFA remains on tools
and could contaminate fresh or live material.
3. 4% phosphate-buffered PFA is stable for ~2-3 days and should
be made >1 day prior to use, if possible.
4. For fragile tissue that is collected directly onto slide after sec-
tioning, modifi cation to accommodate slide-affi xed tissue is as
follows:
After cryo-sectioning, dry the tissue overnight.
4.2. Tips
●
Remove OCT by rinsing ~1-2 min PB and then four dips
●
dH
2
O. Dry the slides overnight.
Apply a hydrophobic barrier:
●
-
Circle the tissue on the slide with a hydrophobic IHC
pen.
Dry at 60°C for 15 min
-
-
Rehydrate:
Remove and cool at RT for 10 min
●
-
100% EtOH 5 min
-
95% EtOH 5 min
-
70% EtOH 5 min
-
Conduct GFAP-IHC as described above for free fl oating
0.1 M PBS 2× 5 min
●
sections
5. Fluorescent signals achieved with diolistic labeling using DiI
and DiO are more susceptible to photo-bleach than GFP or
many other fl uorescent tags on secondary antibodies. Care
should be taken to protect tissues from light and to image
under conditions to reduce photobleach (i.e., lowered confo-
cal laser power and increased photodetector gain).
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