Fos immunohistochemistry staining
Aurélie 2006
Mice c-Fos immunohistochemistry staining protocol
I. Preparation of tissue for immunohistochemistry
Perfusion procedure and brain extraction
On the morning of the perfusion (or day before) make fresh paraformaldehyde (PFA).
Administrate the drug to the mice (THC, 10 mg/kg i.p.) and test for behaviour (or not) 30 min after the injection.
Perfuse (1.5 to 2 hours after the injection) intracardially with 0.9% saline (PBS for Glenn) followed by 4% PFA.
After the mouse is perfused, extract the brains and drop into marked yellow-topped container filled with PFA in PB overnight.
0.9 % Saline
18 g NaCl in 2 L of MQW
Phosphate buffered saline: 0.1 M PBS (last 1 month at RT or in the fridge)
For 1l:
- 10.9 g of di-sodium hydrogen orthophosphate anhydrous (Na2HPO4)
- 3.4 g of sodium dihydrogen orthophosphate monohydrate (NaH2PO4 H2O) or 3.8 g if dihydrate
- 9 g of sodium chloride/L (0.9 %) for perfusion, or not if making PB
4 % paraformaldehyde (PFA) overnight
For 1 L of 4% PFA in 0.1 M PBS:
MIX ON THE DAY BEFORE USE!!
- Heat 600 ml of mQW in a glass beaker to 40-50 °C
- Reduce temperature to low setting and wait for the metal plate to cool a bit, use a thermometer to check when the solution is 40 °C
- Then slowly add 40 g of paraformaldehyde: 20 % at a time stirring constantly, waiting for it to dissolve and watch temperature
- Add 1 ml of 1N sodium hydroxide (1N = 4g/100ml) or a few NaOH pellet to clear
- Place in fridge for 1 hour to cool
- Filter thought whatman #1 (better to separate in 1l bottle to reduce filtering time)
- Add PBS to 1 L
0.05 % Thimerosal PBS 0.1 M (up to 3 months)
1% = 1 g/100 ml so 0.5 g Thimerosal / L of PBS
30 % sucrose PB (no NaCl) (72 h before slicing the brains, but ok for 1 month)
300 g sucrose in 1 L of PB can be store in the fridge
- Place brains in cold 15% sucrose overnight/until they sink
- Place brains in cold 30% sucrose for 2-3 days/until they sink (brains should then be cut within the next week
Place brains in a cryostat with lots of OCT compound on bottom and around the edges and top for at least 1 hour (add OCT on top every 10-20 min until the brain’s frozen) at -11-17°C before slicing.
Slice at 40 µM.
Place serial sections one at a time in 3 yellow-topped 50 ml containers filled with PB.
Store tissue for immediate immunohistochemistry in the fridge (4°C) and the back-ups in freezer (-20°C) in the freezing solution.
Freezing solution:
For 1l in PB (stored in the fridge):
- 300 mls of ethylene glycol.
- 250 mls of glycerol.
When need, place brain slices into 25 mls of this solution in a yellow-topped container and place in (-15 to –20 degree C freezer (NOT –50 or-80 degree C as these are too cold).
Prior to immunohistochemistry, take out of freezer, let stand for 20 min at room temperature, wash at least 3 times in PB for 30 min each before starting step 1 (hydrogen peroxidase).
II. Immunohistochemistry
On day 1, times are not critical, it is at least 30 min, but ok (especially for the NHS) if longer.
For day 2, BAD and glucose oxidase are critical steps.
Day 1 (2-3 hours): All washes and incubations are done at room temperature on an orbital shaker unless stated otherwise.
Note I do not use sodium chloride with the phosphate buffer in this process so it is PB, not PBS.
For most of the steps described below, free-floating sections are transferred from one step to the next using a tea strainer to catch tissue. Old solutions are poured into a 500 ml beaker, the strainer is tipped upside down over the empty 50 ml container and a new solution is poured over the top washing the tissue off the strainer back into the new solution. For other steps, the tissue is gently brushed into a ball using a camel hair paintbrush then the “tissue ball” is gently dropped into a 20 ml glass vial containing primary or secondary antibody or Extravidin.
1) 3% (30% w/v) hydrogen peroxide: 30 min
If using slices from the freezing solution, wash x3 in PB
Add 3 ml of H2O2 for each 97 ml PB
This step reduces background staining of peroxidase (little star-like marks you don’t want).
No washing, ok if bubbles and foam
2) 3% normal horse serum: 30 min
3 ml normal horse serum per 97ml PB to make 100 mls.
No washing
3) Primary antibody 1:10 000: 3 days
(the c-fos antibody can be stored and used for 1 year max)
In glass vials!
3 ml/brain in PBH (phosphate buffered horse serum):
PBH:
For 100 ml in PB:
- 0.1% (100 mg) bovine serum albumin (Sigma A9647)
- 0.2% (0.2 ml) Triton X-100
- 2% (2 ml) normal horse serum
Note: make enough PBH for primary/secondary/Extravidin steps (x3 and store in the fridge for Day 2).
Prepare the solution for all the brains (3 ml per brain) in a glass beaker: c-fos primary antibody 1:10 000 in PBH, and let stand for a few minutes
Add 3 ml (2 ml can be used for mice) of this solution to each glass vial before adding brain
Add the brain (tissue is gently brushed into a ball using a camel hair paintbrush then the “tissue ball” is gently dropped into the 20 ml glass vial containing the primary antibody in PBH).
Place the glass vial into the 50 ml plastic container
Place on shaker for 30-60 min, then place in fridge at 4°C for 72 hrs for c-Fos.
Day "2". Time to complete, 6-8 hours
4) PB wash: 30 min
Drain the slices in a sieve, place in large yellow container filled with PB, incubate on shaker for 30 min.
5) Secondary antibody: 1 hour
Add 2nd antibody as above for primary; eg, for 30 brains (3 mls per vial); you will need 90 ml PBH add 180 uL of secondary antibody for a 1:500 solution. Decant 3ml into each clean glass vial. Pour out the PB into a waste beaker using a strainer to collect the brain slices, then roll tissue into a ball using a fine paint brush and drop the “ball” into the vial and close the cap. Place the glass vial into the 50 ml plastic container
Ensure that you use the appropriate secondary antibody:
anti-rabbit for Jun B, Zif & c-fos (Santa Cruz)
Place secondary antibody vials on shaker for 1 hour.
6) PBS wash: 30 min
7) Extravidin: 1-2 hours.
In the clean vials add 3 mls of PBH, and
1:1000 (3 ul) of ExtrAvidin peroxidase (Sigma) (1 uL/ml)
use batch methods; ie 90 microliters/90 ml PBH for 30 brains
Drain the slices in sieve, form a ball then drop into vial containing 3 ml of solution mixed as above.
Place on orbital shaker for 1-2 hours. (lunch break here).
8) 3 x PBS washes 30 min each
Transfer tissue to the yellow containers with 30-40 mls PBS.
Wash 3 times in PBS for 30 min each on orbital shaker. During this time mix-up the fresh DAB mix.
9) DAB: 20 min
Wear gloves/face mask and goggles when weighing DAB powder
To make DAB mix: you need 20 mls/vial,
- To make enough for 10 vials (200 mls).
- ADD 100 mg DAB tetra hydrochloride (Sigma) to 100 ml PB in a beaker, stir or sonicate for 10 min
- Add 2 mls of 0.4% ammonium chloride (400 mg/100 mls)
- Add 2 mls of 20% D-Glucose (Sigma) (20g / 100 mls).
- Add 2 mls of 2% nickel ammonium sulphate (2g / 100 mls).
- Stir until DAB (pinkish solution) is dissolved.
- Then filter through Whatman #1 paper.
- Pour into a graduated cylinder, add PB until you get the required 200 ml (20 mls per brain),
- The clear solution is ready for use.
- Transfer washed sections into 20 mls DAB mix per vial,
- Place on orbital mixer for 20 min
Note: wash all glassware and instruments with bleach that came into contact with DAB
10) Glucose oxidase: 5-20 min.
To visualize DAB reaction:
- Pipette 20 ul of glucose oxidase into each vial.
- Time this reaction with a stopwatch as this is a critical step in the procedure, usually 5-10 min for c-Fos; 10-15 for TH. WE now use a 10 min time for all c-fos staining
- To stop the reaction: drain slices and then place in PB for 30 min then. Wash again.
- Store in fridge until you coverslip or double label
III. Cover-slipping and counting
Gel-coated slides:
- Heat 450 mls of distilled water in a beaker to 50 °C
- Turn off the hot plate, drop in a magnetic stirrer
- Slowly add 5 g of gelatin, and stir until it dissolves (1 hr)
- Add 0.25 g of chrom potassium sulphate dodecahydrate
- Cool to room temp by placing in fridge for 30 min
- Filter through #1 Whatman paper
- Add mQW to 500 ml
Dip each slide twice in the solution and lean against inside a cardboard box with paper toweling on floor of box.
Cover the top of the box with paper toweling to stop dust.
Let stand 4 hr or overnight. Place dry slides in microscope box marked, clean gel-coated slides. Use these when mounting tissue.
Mount slices from PB onto gel-coated slides (takes 1 hour per mice)
Ensure to brush sections flat onto glass, don't leave folds or flaps in the tissue because these weak areas can ruin a good picture or the tissue may even fall off prior to cover slipping.
Make sure you clearly label the tissue on the frosted edge in pencil with mouse's number, type of staining (fos,zif,jun etc) and slide number in serial order A, B etc for identification later.
Dry overnight (or more) before cover slipping.
Clearing:
- Place mounted slides into tap water for 30 sec (this washes off the PB salt) and then,
- 1 min each in 70% alcohol,
- 1 min in 95% alcohol,
- 1 min in 100% alcohol (2 washes),
- Wash in xylene 5 min,
- Then place in clean xylene for 1-2 hours (this can be overnight).
Coverslip and storage of slides (1-2 days for 200 slides)
Ensure there are no trapped air bubbles by tapping on the coverslip with a Q-tip.
Let stand overnight, and then place them in box in descending order (olfactory bulbs to spinal cord) for each mice.
Counting: (Consult Rat brain atlas of Paxinos and Watson, 1986, 1997)
Labelling each glass slide helps this task; it should contain, rat number (eg, 302), slide (A-Z), and tissue section. I normally count from the frosted edge in columns. So that I know that I counted 102 cells in the nucleus accumbens at +0.7 mm, by lining up the reticule at x20 objective, using particular boundary for rat 276, slide C, section 4.
Manual counting using a graticule
You can count Fos or other immunoreactive cells at a specified objective (x10 or x20) by placing an optical grid over the region of interest and counting all filled oval shapes that fall within the grid. Keep a note on what you are actually counting, e.g. All levels of density (light brown, dark brown, or black cells) and filled ovals, partial ovals etc. While counting, move the stage focus up and down to increase the depth of field. This helps to separate overlapping neurons as well as ovals slightly out of focus. Do the counting blind to the treatment (drug) given.
Keep a score sheet with you, like a spread sheet to mark down the number of reactive cells. Do this in a quiet room to reduce distractions and take frequent breaks to decrease fatigue. Even if you choose to use the automated procedure (see below), you should check between the two methods to ensure there is good agreement.
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