Methodology for Imunnohistochemistry

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Methodology for Imunnohistochemistry
A staining process for identifying the proteins
location in cells, tissues by using
antigen-antibody property. Immuno means
antibodies which are used to bind the histo means
tissue sample for detection.

• Related Los Sectioning, Antigen-Antibody
  property
• gt Prior Viewing IDD31. MALDI data analysis
• gt Future Viewing IDD33. Western blotting
• Course Name Immunohistochemistry
• Level(UG/PG) PG
• Author(s) Dinesh Raghu, Vinayak Pachapur
• Mentor Dr. Sanjeeva Srivastava

The contents in this ppt are licensed under
Creative Commons Attribution-NonCommercial-ShareAl
ike 2.5 India license
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Definitions and Keywords
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1. Immunohistochemistry is a method for identifing
   the presence and locating the proteins of
   interest in tissue sections.
2. Antibodies are very specific, recognize the
   targets and binds to protein of interest.
3. Fixing the process by which the cut sections are
   fixed on to the slides with the help of fixative
   solution like paraformaldehyde or formalin.
   Formation of methylene bridge, helps tissue to
   get fixed to the slides. Fixing is the important
   step in imunnohistochemistry.
4. Antigen retrievel the excess formalin used for
   tissue fix needs to be removed before staining
   step. The formation of methylene briddge mask
   antigen sites, this bridge is broken down by heat
   treatment.
5. Microtome instrument used for sectioning of
   tissues.

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Learning objectives
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• After interacting with this learning object, the
  learner will be able to
• Perform cutting and mounting of the sections
• Define rehydrating of the sections
• Choose antigen retrieval process
• Carry out immunohistochemical staining process
• Infer the sections under microscope
• Assess the troubleshooting steps involved in the
  experiments.

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Master Layout
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Fixing the tissues (Slide5-6)

• Cutting into sections (Slide7-8)

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• Rehydrating the sections (Slide9-10)

• Antigen retrieval (Slide11-12)

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• Staining of sections (Slide13-17)

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• Dehydrating the sections (Slide18)

• Microscope viewing (Slide19-20)

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Display the steps along with images next to them
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Step 1

• T1 Fixing the tissues

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3
Show a small piece of pink color like substance,
user should keep the cursor to know it as
tissue.The tissue must be placed in the
petriplate. Now let user pick up fixing solution,
pour into the plate till the tissue is completely
covered with solution. Let user close the
petriplate lid. Keep the setup for18-24hrs at
room temperature. Animate a clock
The tissues can be obtained from the hosipital,
fixing is the important step, depending upon the
tissue size the fixation time changes but ideal
is around 18-24hrs. The fixation process helps
the cells to fix, with the help of fixative such
as neutral buffer formalin or paraformaldehyde
solution available from the vendors.
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Step 1

• T1 Fixing the tissues

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2
tissue blocks
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Instruct user to take out the tissue from
petriplate and place it in the tissue blocks.
Place the tissue exactly in the middle of the
block. Let user click on paraffin solution and
take it and add paraffin solution to the blocks.
Now for a time period, make the paraffin solution
getting solidified. Display the block embedded in
paraffin like shown in the figure above. Please
re-draw the figure.
The fixed tissue must be embedded in paraffin
wax. The tissue blocks with paraffin wax can be
used for tissue embedding. This tissue block will
provide support and paraffin will hold the tissue
at the time of sectioning.
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Step 2

• T2 Cutting into sections

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2
microtome
embedded tissue block
tissue blocks
3
Let user take the embedded tissue block next to
microtome instrument. Let user place the tissue
block onto the microtome. Let user start the
sectioning the tissue, by setting 5microns
thickness parameters. The sectioning can be
carried out manually. Animate user making a
clockwise movement on the handle, with tissue
block moving vertically up and down and sections
coming out in the form of film.(show like thin
white color plate like thing with pink tissue
sections in the middle)
The embedded tissue block must be placed onto the
microtome. User must define the sectioning
thickness.
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Step 2

• T2 Cutting into sections

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2
Tissue section on slide
water bath
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Instruct user to pour water and set the
temperature to 35C of water bath. Let user takes
out the tissue sections from the microtome with
help of forceps. Transfer the sections into water
bath. Animate the sections floating on the water
bath, let user with help of forceps cut the
sections. Once the sections expands, let user
take a slide, dip in water bath to take out the
sections on the slide. Now keep the slide at room
temperature for drying. Display the final figure
of tissue section on the slide. Events must
happen when the user clicks on it
Once the sections are produced from microtome,
they must be transferred on to water bath, so
that the paraffin wax gets melted and sections
are expanded. Now take the section exactly in the
middle of the slide. Tissue sections must be
mounted on positive charged slides.
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Step 3

• T3 Rehydrating the sections

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100 ethanol
70 ethanol
30 ethanol
Ethanol
11 Xyleneethanol
95 ethanol
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50 ethanol
Xylene
Water
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Audio Narration (if any)?
Description of the action/ interactivity
Instruct user to take out ethanol, xylene and
water bottle from the rack and keep it on table.
Instruct user to prepare 11 xylene and ethanol
solution, let user take the xylene bottle and
pour to measure 10ml and follow same for 10ml
ethanol, mix and transfer the solution to 11
bottle. Let user measure 20ml ethanol and
transfer it to 100 ethanol bottle. Now for 95
ethanol, let user measure 9.5ml of ethanol and
make up to 0.5ml by adding water(done by setting
the pipette to 500ul and take water to add to the
ethanol). Similarly for 70 (7ml ethanol3ml
water) 50 (5 ml ethanol 5 ml water) for 30(3ml
ethanol 7 ml of water) prepare accordingly and
transfer to the bottles.
The ethanol and xylene solution must be prepared
for the rehydrating the sections. The ethanol
must be prepared in decreasing concentration of
grades.
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Step 3
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T3 Rehydrating the sections
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3min
3min
3min
2X3min
3min
3min
3min
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Instruct user to take out the slide which is kept
for drying using the forceps . Now dip the slide
in 100 xylene solution for 3min for 2 times.
Animate for 4min to take out the slide and keep
it in 11 bottle for 3min. From there let user
transfer the slide to decreasing alcohol grades
for 3min each. Animate user taking the slide from
each bottle and place in the next bottle by user
control. After last step of 30 ethanol, let user
place the slide in distilled water. Please
re-draw the figure.
The xylene solution treatment helps to remove the
paraffin wax and alcohol treatment rehydrate any
water content from the tissue sections. This step
exposes the binding property of the cells.
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Step 4
T4 Antigen retrieval
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EDTA buffer
EDTA
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3
Instruct user to prepare EDTA buffer. Let user
take out EDTA, water bottle from the rack. Let
user takes a paper, tare it over the balance to
weigh 0.037g, transfer it to beaker, to this let
user measure 100ml of distilled water and add to
the beaker. Let user shakes the bottle to
dissolve the powder into the solution. Animate
powder getting into the solution and user
labelling the bottle as EDTA Buffer.
Prepare 1nm EDTA buffer, it is used in antigen
retrieval step. For tissue for higher pH user
need to adjust pH or u can use Tris-EDTA of pH
9.0 also
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Step 4
T4 Antigen retrieval
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EDTA buffer
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coupling jar
Oven
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Instruct user to take out slide from the water by
forceps, with help of tissue let user take out
the excess water from the slide. Transfer EDTA
buffer to coupling jar, around 20ml. Now place
the slide into the jar and keep it in microwave
Oven. Let user set the temperature and time for
oven. Animate user control to set the temperature
for 40C for 15-20min. During heating animate the
buffer getting boiled, instruct user to stop the
oven. Let user take out the jar from oven,
removes the slide and place it in water jar.
Use sufficient volume of buffer in order to cover
the slide. Be sure to watch for evaporation and
for boiling to avoid slides from dry out. Less
time may leave the antigens under-retrieved and
more time may leave them over-retrieved, user
must take a call. Now user can proceed to
immunostaining step.
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Step 5
T5Staining of sections
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Sodium azide
TBS
Tris
Nacl
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3
Instruct user to prepare TBS (Tris buffered
saline), let user take out tris free base, NaCl,
sodium azide and distilled water from the rack
and place it over the table next to balance. Now
let user tare the balance with paper, take out
spatula to weigh 0.61g of Tris, 0.88g of NaCl and
pipette out 5ml of sodium azide, transfer all
these to beaker. Let user measure 90 ml distilled
water and pour to the beaker, to mix everything
into the solution. And transfer to the measuring
cylinder and reading should be 95 ml ,click on
water to add to the cylinder to make to 100ml.
Let user label the bottle as TBS.
Use sufficient volume of buffer in order to cover
the slide. Be sure to watch for evaporation and
for boiling to avoid slides from dry out. Less
time may leave the antigens under-retrieved and
more time may leave them over-retrieved, user
must take a call even with the pH of TBS buffer.
Now user can proceed to immunostaining step.
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Step 5
T5Staining of sections
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TBS
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primary antibody
Secondary antibody
working stock
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Instruct user to dilute BSA, primary and
secondary antibodies. let user take out the
primary and secondary antibody from the -20C
freezer, keep it on ice for 5min to thaw, now set
the pipette to 1ul, take out the 1ul of primary
antibody, transfer it into a fresh tube. To the
fresh tube add 1ml of TBS, vortex it properly,
user label it as working stock. Animate user
preparing for secondary antibody and BSA with
similar steps. Place back the antibodies back to
freezer. Events must happen as and when the user
clicks on the hand image.
The concentration of working stock depends on
user experiment. The working solution must be
prepared in TBS depending on the concentration
required for staining.
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T5Staining of sections
Step 5
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Let user take out humidifier chamber, wet some
tissue with water, place the wet tissue on the
sides of chamber. Instruct user to take out the
slide from the water jar, with tissue let user
take out excess water from the slide. Let user
add 500ul of BSA with pipette onto the slide,
keep it on humidifier chamber for 2hours
incubation at room temperature. Now take out the
slide, wipe around the section with tissue paper.
Now let user apply 500ul of primary antibody on
to the slide and keep it in chamber for overnight
at 4C freezer. Animate the pipetting action as
per the instructions
The sections must be treated with BSA to block
unspecific binding, later add primary antibody
and place it for incubation for overnight at 4C.
Incubation must be done in humidifier chamber to
avoid drying of sections.
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T5Staining of sections
Step 5
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2
1hours
3
Let user take out slide from humidifier chamber,
do wash with TBS for 3 to 3 times. Instruct user
to take out the slide from the TBS jar, with
tissue let user take out excess TBS from the
slide. Let user add 500ul of Secondary antibody
with pipette onto the slide, keep it on
humidifier chamber for 1hours incubation at room
temperature.
After primary treatment, wash the slides with TBS
to remove unbound primary antibodies. User can
even go for TBST buffer for better result. Now
after addition of secondary antibody incubate for
1hour depending upon the experiment requirement
and the concentration required for secondary
antibody.
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Step 5
T5Staining of sections
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Stain
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3
Let user take out slide from humidifier chamber,
do wash with TBS. Instruct user to take out the
slide from the TBS jar, with tissue let user take
out excess TBS from the slide. Let user add 500ul
of Stain with pipette onto the slide, keep it on
humidifier chamber for 2-3min.
The staining process depends on the user
requirement, tissue used and the setup of
microscope. Commonly used counterstains are
hematoxylin (blue), nuclear fast red or methyl
green. Once staining is done the slide must go
through dehydration step before viewing under
microscope.
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T6 Dehydrating the sections
Step 6
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2
3min
3min
3min
3min
3min
3min
3min
3
Instruct user to take out the slide which is kept
on humidifier chamber. From chamber let user
transfer the slide to increasing alcohol grades
for 3min each. Animate user picking the slide
from each bottle and place in the next bottle by
user control. Now dip the slide in 100 xylene
solution for 3min, followed by 11 bottle for
3min and final wash in 100 xylene. After xylene
wash, take out the slide, remove excess liquid
from the slide with tissue paper. Please re-draw
the figure.
The dehydrating step is carried out to remove
excess stain if any present in the slide.
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Step 7
T7 Microscope viewing
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Mounting solution
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3
Let user take out mounting solution and cover
slip from the rack and place it on the table. Let
user take out mounting solution around 10ul with
the pipette, and drop it over the slide. Now let
user take out the cover slip, place it over
slide, the cover slip must cover the tissue like
shown in the figure.
The mounting solution like organic mounting media
have better refractive index than aqueous
mounting media. Now the slide is ready for
viewing under microscope.
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Step 7
T7 Microscope viewing
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Let user take the slide and mount it on
microscope, let user ON the microscope. Let user
do the controls over making adjustment on keeping
the side on the platform, placing the clips over
it, adjusting the focusing, fine tuning of
zoom-in, zoom-out. Animate the user adjustment
with change in appearance of the display. Display
the final image of tissue, once user is done with
adjustment.
For better image, do proper adjustment and fine
tuning on the microscope. Once the final image
comes up, save the image. The confirmed protein
expression images can be published in the
research articles.
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Slide 7-8
Slide 11-12
Slide 13-17
Slide 18
Slide 9-10
Slide 4-6
Slide 19-20
Slide 1-4
Introduction
Tab 01
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Name of the section/stage

• Animation area
• In slide-16 depending upon user input for
  antibody, five some options for user to select
  the secondary antibodies.
• Instruction if the primary antibody is raised in
  rabbit, then the secondary antibody could be goat
  anti-rabbit provide options accordingly.

Interactivity area
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1
1. What is the meaning of Histo? A) Antibody
B) Tissue C) Antigen D) All
of above 2. For better and sharper microscopic
image, use? A) Xylene. B) ethanol. C) DHAPI. D)
Organic mounting media.
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Questionnaire
APPENDIX 1

• 3. Hematoxylin stains?
• Blue color
• Red color
• Green color
• Yellow color
• 4. Antigen retrieval step breaks?
• A) methylene bridge
• B) Glycine bidge
• C) Alanine bridge
• D) Glutamine bridge

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Questionnaire
APPENDIX 1
Question 5
BSA wash is given to block?
a) Non-specific binding b) Specific binding c)
Methylene binding d) None of above Answer a)
Non-specific binding
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APPENDIX 2

• Links for further reading
• www.abcam.com/technical
• Books
• Proteomics A cold spring harbor laboratory
  course manual by Andrew J L and Joshua L, 2009.
• Immunocytochemistry a practical guide for
  biomedical research by Richard burry, 2010.

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APPENDIX 3
Summary
To study the section, main step is to optimize
the antibody concentration required for specific
binding and observation to check the paraffin
sections/tissues. Use of antibodies with all
stepwise protocol helps to obtain better result.
Some of the stains are harmful in nature, need to
used under proper and safe laboratory protocols.
Drying of slides need to avoided during the
process.