Immunohistochemistry of tight junctional proteins

(ZO-1/ocludin) in Caco-2 cell monolayers

1) Caco-2 cell monolayers cultured on Transwells (Corning Costar, Cambridge, MA)

for at least 21 days in the MEM. Monolayers showing TEER values above 600
selected for further experiments.
2) NPs treatment
a) Untreated monolayers
b) NPs Treated monolayers: 30 min, 60 min, 90 min, 120 min.
c) Recovered monolayers: 6 h, 12 h and 24 h post treatment.
3) Wash monolayers with PBS and fix them using freshly prepared 3.75%
paraformaldehyde solution. Wash with PBS
4) Permeabilize cells for 30 minutes at 37℃ using 0.2% Triton-X-100 and 10ug/ml
RNAse in PBS. Wash with PBS 3 times.
5) Incubate with 5% blocking serum in PBS for 1 hour at 37℃. Don’t wash
6) Incubate with primary antibody (mouse anti ZO-1 / mouse anti-occludin)
diluted 1:50 in 5% blocking serum in PBS for 1 hour at 37℃. Wash with PBS 3
times (5 min each)
7) Incubated in with biotinylated secondary antibody (horse antimouse IgG)
diluted 1:100 in 5% normal goat serum in PBS for 1 hour at room temp.
8) Make ABC solution at least 30 min before use.
9) Wash samples with PBS 3 times (5 min each)
10) Incubate in ABC solution (20 µl each sample) for 30 min.
11) Wash with PBS 3 times (5 min each)
12) Make DAB solution, follow instructions given on Kit.
13) DAB reaction, 2 min (check under microscope to stop reaction with PBS)

Primary Antibody: mouse anti-human ZO-1, anti-occludin

Secondary Antibody: Horse antimouse IgG (biotinylated)
Blocking Serum: a. should not crossreact with primary and secondary AB
b. the species must be same with secondary AB (Horse serum)

ABC solution: Avidin Biotinyl peroxidase complex (40 µl A+ 40 µl B in 4ml PBS)

DAB solution: 1 ml DI, 20µl Buffer + 40µl DAB + 20µl H2O2

 Number of Caco-2 monolayers
ZO-1 Ruthenium Red Occludin
Untreated 4 4 4

NPs Treated
30 min 2 2 2
60 min 2 2 2
90 min 2 2 2
120 min 2 2 2

Recovery
6h 2 2 2
12 h 2 2 2
24 h 2 2 2
Total 18 18 18
Grand Total 54
 Sample name meaning

1 Control ZO-1 Control monolayers (without NPs treatment) stained for

ZO-1
(A)/(B)/(C)/(D)
(n=4, A-D)

2 Control Occludin Control monolayers (without NPs treatment) stained for

Occludin
(A)/(B)/(C)/(D)
(n=4, A-D)

3 Control Ruth Red Control monolayers (without NPs treatment) for

Ruthenium Red Staining
(A)/(B)/(C)/(D)
(n=4, A-D)

4 Recovery ZO-1 Cell monolayers recovered after NPs treatment , at

different time intervals (6h/ 12 h/ 24 h ) and stained for
6h/ 12 h/ 24 h
ZO-1
A/B
(n=2, A&B)

5 Recovery Occludin Cell monolayers recovered after NPs treatment , at

different time intervals (6h/ 12 h/ 24 h ) and stained for
6h/ 12 h/ 24 h
Occludin
A/B
(n=2, A&B)

6 Recovery Ruth Red Cell monolayers recovered after NPs treatment, at

different time intervals (6h/ 12 h/ 24 h ). For Ruthenium
6h/ 12 h/ 24 h
Red staining
A/B
(n=2, A&B)

7,8 NPs ZO-1 / Occl. NPs treated cell monolayers, stained for ZO-1 or
Occludin
30 min/ 60 min/ 90
min/ 120min 30 Min to 120 min -> duration of NPs treatment
A/B N=2 , A &B

9 NPs Ruth Red NPs treated cell monolayers for Ruthenium Red staining
30 min/ 60 min/ 90 N=2, A&B
min/ 120min
A/B