Human/rat brain microvessel isolation

(alternatively also used for small samples)

Preparation for the experiment

1.

2.

sterilization (if heat sterilization is not possible, use autoclave for everything):
heat serilization - 3h 160 C :homogenisation tube, scissors, forceps, scapels, filter paper,
petri dish
autoclave - 120 C: 50 ml and 15 ml conical and plastic tubes
20 min ethanol: the screw of the homogenisator, than let it to dry
Ringer-Hepes buffer
150 mM
2.2 mM
0.2 mM
5.2 mM
2.8 mM
5 mM
6 mM

NaCl
CaCl2 2 H2O
MgCl2 6 H2O
KCl
glucose
Hepes
NaHCO3

1L
8.7 g
0.323 g
0.0406 g
0.387 g
0.504 g
1.19 g
0.504 g

500 mL
4.35 g
0.1615 g
0.0203 g
0.1935 g
0.252 g
0.595 g
0.252 g

pH 7.4, sterile if possible

3.

1%BSA-RH: 1g into 100 mL RH, filter with sterile filtered

4. 17.5% Dextran in Ringer-Hepes:

64-76 kDa average mol.weight dextran (Sigma, D4751):
17.5 g into 100 mL RH, sterile filtered (little bit viscous)
4.

use refrigerated centrifuge. The G values has to be calculated according to the radius
of the rotor.
In the Deli lab: the used Eppendorf centrifuge is in the 238. laboratory, where 400g for
washing is 2000rpm. No refrigerated centrifuge is used.

The experiment:
Before the experiment rinse all the glassware, plastic ware with 1%BSA-PBS to avoid the
attachment of the microvessels to the surfaces; also always rinse the pipette tips!
The beginning of the protocol is the same, as for the primary endothelial cell isolation.
1. Deeply anesthetize the rats and mice (dry ice) OR use cervical dislocation.
2. Rinse the head of the animals 1x in 70% ethanol, then Betadine (cc. iodine solution)
+alcohol, then cut the heads with scissors and put into a big Petri dish on ice.
3. Cut the skin with a pair of small scissors, and fix the skin edges to the dissecting pad
with needles. Use another pair of scissors for cutting the bone with a sagittal incision.
Keep the scissors in ethanol when not in use. Open the skull with the curved forceps and
take the forebrain and put it to a dish containing PBS on ice. (we do not use the
cerebellum and the bulbus olfactorius)
4. Put sterile chromatography paper into a sterile big dish. Take off the meninges by
spreading half a brain on the paper and gently rolling. Remove also the white matter and
choroid plexus if possible. Put the tissue pieces into a sterilized glass dish (60 mm)
containing 1 ml Ringer-Hepes (RH) on ice. Weigh the samples on a sterile aluminum
foil.
5. Cut the tissue to very small pieces (1 mm3) with scalpels in the 1 mL RH.
6. Transfer the cut brain cortex samples to homogenizers on 4C in Ringer-Hepes (RH)
solution, homogenization: 4 mL of ice-cold RH / g of tissue (min. 25 times); 1 rat brain
is around 0.8-0.9 g after the removal of the meninges.
7. The resulting homogenate is centrifuged at 1000 g for 10 min in RH in 15ml tube
8. The microvessel enriched pellet is suspended in 10mL 17.5% dextran (prepared in RH)
(64 76 kDa, Sigma) and centrifuged for 15 min at 1500 g at 4 C (in 15ml tube)
9. The resulting pellet is suspended in 2mL RH containing 1% BSA, while the supernatant
containing a layer of myelin is centrifuged twice more for 15 min at 1500 g at 4 C.
Collect the supernatant all the time into another tube, suspend the myelin again before
centrifugation! The three pellets are pooled (6 ml altogether after the 3rd round).
10. At the end filtration of the pooled pellets are done with the PluriStrainer mesh:
yellow: 100um mesh
green: 20um mesh
What flows through is the debris and capillary:

Then the strainers are turned and macrovessels (100um) and microvessels (20um) retained by
the mesh are washed with 15ml buffer, and centrifuged for 10 min, 1000 g, 4C

11. Then everything is 1x washed again in 10mL RH at 700g, 5min 4C;

12. Then the pellets are collected in eppendorfs with 1000g, 5min
13. The pellet can be frozen at -80C or used immediately for Western Blot or RNA
isolation, immunohistochemistry.

Microvessel immunohistochemistry
Deli lab, 2015

Solutions
Dulbeccos PBS (1x)
1L
KCl
KH2PO4
NaCl
Na2HPO4 x 2 H2O

0,2 g (2,7 mM)

0,2 g (1.5 mM)
8 g (0,136 M)
1,424 g (6.5 mM)

+ Millipore Water (TDW)

pH 7.4
PFA solution
3% PFA in PBS; 3g in 100 mL
Have to be mixed with magnetic stirrer and heated (max 60C); and add a drop of 1M NaOH.
At the end, it has to be cooled and the pH set to 7.4.
Also filter it with sterile syringe filter or filter paper.
3%BSA-PBS: 3g into 100 mL PBS, sterile filtered (BSA: A7906, Sigma)

Experiment:
In an eppendorf tube:
For fixation + block + wash 500 l / eppendorf volume is used. We centrifuge the eppendorfs
with the cells all the time at 400g; 2min (at our eppendorf centrifuge it is 2000rpm).
At the end of the isolation pellet is collected with 700 g; 5 min in 1mL of buffer.
1. Fixation of the cells in 4% PFA-PBS; 30min, RT
2. Wash the cells 2 times in 0.3% BSA-PBS
3. Permeabilization in 0.2% TX-PBS; 15min 4C
4. Wash the cells 2 times in 0.3% BSA-PBS
5. Block the cells in 3% BSA-PBS; 30min, RT
6. Add the first antibody, for us:

rabbit anti - ZO-1: 1:200 (Invitrogen, 61-7300);

rabbit anti-Cl-5: 1:200 (Sigma, SAB4502981)
rabbit anti--catenin 1:200 (Sigma, C2206)
rabbit anti - occludin: 1:50 (Invitrogen, 71-1500)
in 3% BSA-PBS; 1.5 hours; 4C in 300 l

7. Wash the cells 2 times in 0.3% BSA-PBS

8. Add the secondary antibody, for us:

Anti rabbit CY3: 1:400 (Sigma, C2306)

+ H33342 nucleus staining in 1000 x dilution
(Calbiochem, 382065)
in PBS; 50min; RT, in 300 l

9. Wash the cells 2 times in 0.3% BSA-PBS

10. Wash the cells once in PBS
11. Wash the cells once in DW
12. Mounting: suspend the pellet very carefully with 50 l mounting medium and put the
50 l onto a cover glass + cover with a glass slide