Transcript Immunohistochemistry ESE3 What is Immunohistochemistry? • a method used to detect and achieve visualization of the presence and distribution of a specific antigen in cells.

Immunohistochemistry
ESE3
What is Immunohistochemistry?
• a method used to detect and
achieve visualization of the
presence and distribution of a
specific antigen in cells or tissues
• uses antigen-antibody
reactions, a ABC reagent and an
enzyme substrate to achieve
staining
What
Did
• stained prostate
tissueI samples
for ESE3
• troubleshooted ESE3 antibody
using the controls
- no antibodies
- primary antibody only
- secondary antibody only
- IgG and secondary antibody
ESE3
• a nuclear protein
• a transcriptional factor
• epithelial-specific expression
• expressed in normal cells
• down-regulated in cancerous cells at both the mRNA and
protein levels
• acts as a tumor suppressor due to it’s involvement in the
control of cell differentiation
Immunohistochemistry
Protocol
Part 1
Deparaffinize and
1. 3 x10Rehydrate
min xylene; 2 x 2 min in 100% ETOH, 2 x 2 min in 70%
ETOH.
Make sure to shake off excess xylene on slide before putting the slides into EtOH in order no
to
dilute the EtOH.
2.
Rinse sides gently with ddH2O squirt bottle being careful not
to
on the
Wash
with
ddH
O
in
Coplin
jar
for
2
1.rinse
Wipedirectly
off excess
ddHsection.
O
from
slide
work
quickly
to
ensure
2
2
x
2
min
on
shaker
at
120
rpm.
that
uenching
Endogenous Peroxidase
section does not completely dry out. To do this shake the
slide
and use a kimwipe to get rid of any extra liquid.
2. Pipette enough hydrogen peroxide (3% H2O2 in H2O) to
cover
the entire section and incubate for 10 min.
Does not need to go into a humidity chamber.
3.
Wash Sections with 1 x TBST with squirt bottle and then
1. While section is incubating in hydrogen peroxide, pre-heat
AntigenRetrieval
Antigen
Retrieval Buffer in food steamer for 15 mins.
Lift up the containers in order to fill the bottom of the steamer up with tap water, up to the
second line.
Put approximately 200 mL of antigen retrieval buffer into the glass slide holder in the
steamer.
Incubate slides in pre-heated Antigen Retrieval Buffer for 20
min.
Use forceps to transfer the slides into the slide holder.
3. Remove lid from the food steamer after the 20 min period and
let
the slides sit inside the steamer for 10 min.
4. Remove the entire slide box with buffer and let sit at room
temperature on bench for 20 mins.
5. Wash slides in 1 x TBST for 3 x 2 min in Coplin jar on shaker
2.
Blocking
and
Primary
Antibody
1. Gently dry slides and put in humidity chamber which is
Addition
tupperware lined with moist paper towels and a slide holder.
Redraw hydrophobic barriers, if they come off.
2. Incubate
slides for 1 hr at room temperature with blocking
buffer in the humidity chamber: 10% rabbit serum (RS) and
1 x TBST.
5 slides
2x[1.00 mL of 1xTBST and 100 uL of RS]
3 or 4 slides
1.25 mL of 1xTBST and 125 uL of RS
3. Wipe
and shake off excess blocking buffer. Add primary
antibody
diluted in blocking buffer and incubate overnight at 4 C in
humidity chamber
5 slides (1:300)
2x[3.33 uL of ESE3 in blocking buffer]
Rat IgG (1mg/mL) - 1:300
1.33 uL of Rat IgG in blocking buffer
3 or 4 slides (1:300)
4.16 uL of ESE3 in blocking buffer
2. Add
appropriate biotinylated secondary antibody to each
Part 2 - Visualization with Vector Labs ABC Kit
section.
Incubate in humidity chamber for 1 hr at room temperature.
For anti- rat biotinylated antibody (1:200)
5 slides
5 uL in 1.0 mL of 1x TBST
3 or 4 slides
6.25 uL in 1.0 mL of TBST
3. During
this incubation, prepare ABC reagent following
manufacturer’s instructions or for a small volume add 12.5 uL
of
Reagent A to 1.25 mL of dilution buffer, TBST, then
immediately add
12.5 uL of Reagent B and mix well. Allow for ABC reagent to
sit at
room temperature for about 30 min.
4. Wash Slides for 2 x 5 min in 1 x TBST on shaker.
5. Incubate sections with ABC reagent for 1 hr in humidity
slides.
Incubate slides in DAB solution for 4 min
In 5.0 mL of distilled H2O
i. Add 2 drops of buffer, mix well
ii. Add 4 drops of DAB stock solution, mix well
iii. Add 2 drops of Hydrogen Peroxide, mix well
8. Wash
slides under running distilled H2O for about 30 sec.
Incubate in distilled H2O for 5 min.
9. Counterstain with appropriate amount of haematoxylin [HE]
for
40 sec. Wash slides under running tap H2O for about
30 sec. Incubate in tap H2O for 5 min.
10. Clear and dehydrate slides: 2 x 3 min 70% EtOH, 2 x 3 min
100% EtOH, 2 x 3 min xylene.
11. Coverslip with Cytoseal mounting medium.
Results
Normal
40x
60x
Cancerous
PIN
Normal Gland (40x)
nuclear staining
Cancerous Gland (40x)
no nuclear staining
PIN (40x)
no nuclear staining
nuclear staining
Special Thanks To
Dr. Tang
&
Judy Yan
supervisor