10. Mount a clean coverslip onto the slides using Vectashield
HardSet with DAPI.
11. Allow the slides to dry in the dark at room temperature for
15 min prior to imaging with a confocal microscope (see Note
4).
3.3.5
MTT Assay
1. Make up the MTT solution with 1 mg/mL solution of thiazo-
lyl blue tetrazolium bromide in phenol-free DMEM.
2. Carefully unclip the Alvetex® membranes and move them to a
fresh 12-well plate.
3. Wash the membranes twice with PBS.
4. Add 1 mL of the MTT solution to each well.
5. Incubate the membranes for 1 h at 37 C.
6. Aspirate the MTT solution and add 1 mL of acidified isopro-
panol to each well.
7. Place the plates on a shaker at 120 rpm for 15 min.
8. Add 180 μL of isopropanol to wells in a 96-well plate.
9. Add 20 μL of the acidified isopropanol to the wells in the
96-well plate.
10. Read the absorbance of the wells at 570 nm using a plate
reader.
Table 1
Primary antibodies for use with immunofluorescent staining
Primary antibody
Dilution
Species
Supplier
Product code
MRP2
1 in 100
Mouse
Santa Cruz
cc-71603
MRP3
1 in 100
Rabbit
Abcam
ab204322
Claudin 2
1 in 100
Rabbit
Abcam
ab53032
α-Smooth muscle actin
1 in 100
Mouse
Abcam
ab7817
E-cadherin
1 in 100
Mouse
BD Biosciences
6011082
Ki67
1 in 100
Rabbit
Abcam
ab16667
Table 2
Secondary antibodies for use with immunofluorescent staining
Secondary antibody
Dilution
Supplier
Product code
Donkey anti-mouse Alexa Fluor 488
1 in 1000
Invitrogen
A21203
Donkey anti-rabbit Alexa Fluor 594
1 in 1000
Invitrogen
A21205
Applying Stirred Perfusion to 3D Tissue Equivalents to Mimic the Dynamic In. . .
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