Immunological Response to Experimental Intracerebral Hemorrhage: Morphological Assessments - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 615

2.7. Recipes

Cryoprotectant
100 ml glycerol (AKA glycerin).
120 ml Ethylene glycol.
180 ml 0.5 M phosphate buffer (solution C).
Phosphate buffer
1 M NaH 2 PO 4 = 69 g add 500 ml ddH 2 O (solution A).
1 M Na 2 HPO 4 × 7 H 2 O = 134 g add 500 ml ddH 2 O (solution B).
0.5 M Phosphate buffer (pH 7.2) = 28 ml solution A + 72 ml solu-
tion B + 100 ml dH 2 O (solution C).
When making solution B, store excess in Pyrex container. Solution
will precipitate, so reheating will be needed.
Phosphate buffered saline (PBS) 0.1 M, pH 7.4
Dissolve PBS tablets (PBS tablets, MP Biomedicals, LLC; cat
#2810305) in ddH2O in proportion 1 tablet to 100 ml
ddH2O. An expected pH is 7.4. Check pH with pH meter;
add KOH or HCl for pH correction, if needed.

4% Paraformaldehyde in 0.1 M PBS (PFA)
To prepare 4% PFA from 32% PFA concentrate (PFA 32%, 100 ml;
Electron Microscopy Science, cat.#15714) take 700 ml PBS
and add 100 ml 32% PFA (1 bottle).
Note : The fi nal volume will be 800 ml, 8 tablets of PBS are needed
to prepare this solution.

Wash buffer
0.05% Tween: 1 L 0.1 M PBS add 500 μ l Tween.

Incubation buffer
0.3%Triton X-100: add 3 ml of Triton X-100 into 1 L of Wash
Buffer.

Equipment
Infrared scanner (Odyssey-system, LI-COR Biotechnology, USA),
Fig. 5a .
Protocol
1. Clean the scanner surface with a distilled water and allow to
dry for several minutes.
2. Wipe slide with 95% ethanol.
3. Place slide up face on the Odyssey scanning surface and close
the lid.
On the scanner console (Fig. 5b )
4. To scan slides, leave the preset “membrane” (#1 in Fig. 5b ).
5. Make sure the check boxes are selected for the dyes you want
to scan. 700 corresponds to IRDye 700 reagents and 800 to
IRDye 800 reagents (#5 in Fig. 5b ).

2.8. ICH Scanning
Procedure