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Blood–brain barrier permeability

Evans Blue Extravasation

Calculate mouse-specific injection volumes, prepare extraction reagents, and generate the complete 96-well fluorescence plate layout before the experiment.

Experiment setup

Define the experimental groups and mouse count first, then generate the dosing table and enter each post-SAH day 1 morning body weight.

Evans Blue dose 4 µL/g 2% EB in saline
Detection 620 / 680 nm Excitation / emission

Step 1

Define groups and mouse counts

#Group nameNumber of mice
1 group · 1 mouse Mouse table is up to date.

Step 2 · Animal dosing

Edit Mouse ID and body weight

Injection volume = body weight × 4 µL/g
#Mouse IDGroupBody weightInjection volume
Automatic planning Each 96-well plate holds the A–H standard curve plus up to 10 mice. Mouse 11 starts a new plate with a fresh standard-curve layout.

96-well plate layout

Columns 1–2 contain standards A–H. Each remaining column contains one mouse: ipsilateral hemisphere ×3, contralateral hemisphere ×3, and serum ×2.

Plates1
Mice1
Standard wells / plate16
Sample wells / mouse8
Standard Ipsilateral hemisphere Contralateral hemisphere Serum 200 µL per well

Preparation calculations

Quantities update with animal count, body weights, plate count, and the selected overage.

Perfusion

Ice-cold 1× PBS

60 mL / mouse
Exact perfusion volume
60 mL
Prepare with overage
66 mL
Circulation time
3 hours

Extraction

50% TCA in 1× PBS

T0699 : PBS = 1 : 1
Brain extraction (800 µL × 2 sides)
1.6 mL
Solution A + standards
17.733 mL
Total work solution + overage
21.266 mL
Sigma T0699 (~100% w/v)
10.633 mL
1× PBS
10.633 mL
Corrosive reagent T0699 is a strong acid. Prepare using the laboratory’s approved PPE, hood, and chemical-safety procedure. The 1:1 calculation uses the nominal ~100% w/v concentration; check the lot COA when exact concentration matters.
Sigma T0699 product information ↗

Blood sample

Serum dilution

1 : 500

For each mouse: 2 µL serum + 998 µL 1× PBS. Load 200 µL into each of two wells.

Total serum
2 µL
Total PBS for dilution
998 µL
Diluted sample prepared
1 mL

Standard curve

Eight concentrations, two wells each

1 mL per concentration
Solution A · 20 ng/µL 10 µL of 2% Evans Blue + 9,990 µL of 50% TCA work solution = 10 mL
StandardFinal concentrationSolution A50% TCA work solutionPlate use
A 2000 ng/mL 100 µL 900 µL 2 × 200 µL / plate
B 1000 ng/mL 50 µL 950 µL 2 × 200 µL / plate
C 800 ng/mL 40 µL 960 µL 2 × 200 µL / plate
D 600 ng/mL 30 µL 970 µL 2 × 200 µL / plate
E 400 ng/mL 20 µL 980 µL 2 × 200 µL / plate
F 200 ng/mL 10 µL 990 µL 2 × 200 µL / plate
G 100 ng/mL 5 µL 995 µL 2 × 200 µL / plate
H 50 ng/mL 2.5 µL 997.5 µL 2 × 200 µL / plate

The original 1 mL recipe covers one or two plates. For three or more plates, the table scales automatically.

Preparation summary & protocol reference

Copy or print the calculated plan. The reference below is informational and does not record experimental execution.

Calculated plan

Experiment overview

Animals
1 mouse
Plate layout
1 × 96-well plate
Circulation
3 hours
2% Evans Blue
120 µL
Cold PBS perfusion
66 mL
50% TCA work solution
21.266 mL

Preparation quantities

Samples and wells

Brain hemispheres
2
Brain wells
6
Blood samples
1
Blood wells
2
Standard wells
16
Total occupied wells
24

Reference workflow

Evans Blue extravasation

Ex / Em: 620 / 680 nm
  1. Prepare and inject.Prepare 2% Evans Blue in saline. Inject 4 µL/g using the post-SAH day 1 morning body weight.
  2. Circulate and collect blood.Allow the selected 3- or 4-hour circulation period, then collect blood.
  3. Perfuse and collect brain.Perfuse with 60 mL ice-cold 1× PBS. Collect the brain, separate ipsilateral and contralateral hemispheres, and record tissue weights.
  4. Homogenize.Add each hemisphere to 800 µL of 50% TCA work solution and homogenize for 100 strokes.
  5. Clarify brain samples.Centrifuge at 15,000 rpm for 15 minutes at 4°C, three times, and collect supernatant.
  6. Prepare serum.Centrifuge blood at 5,000 × g for 15 minutes at 4°C, twice. Dilute 2 µL serum with 998 µL 1× PBS.
  7. Load and read.Load 200 µL per well according to the generated plate map and measure at 620 / 680 nm.