Sep 15, 2022
Brain processing, slicing and immunohistochemistry protocol
- Andrew Hunter1
- 1Northwestern university
Protocol Citation: Andrew Hunter 2022. Brain processing, slicing and immunohistochemistry protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2xrpg5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 15, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70103
Keywords: ASAPCRN
Abstract
This is a step by step procedure from collecting brain samples to immunohistochemical staining and mounting brain slices.
Materials
Reagents, Instruments, and Materials
For 4% PFA in 0.1M PB pH7.4 preparation: (D1)
- Corning stirrer/Hot Plate
- 500mL Fixative-labelled Erlenmeyer flask with stir bar
- 250mL PFA-designated graduated cylinder
- Glass funnel
- Filter paper
- Thermometer
- 1L volumetric flask
- 500mL storage bottle for fix
- Distilled H2O
- Electron Microscopy Sciences Granular Paraformaldehyde
For perfusion fixation
Phosphate Buffered Saline, pH 7.4
- 4% Paraformaldehyde solution (4% PFA)
- WPI SP100I Syringe Pump
- Steel surgery bin
- Surgical tools
- (scissors, scalpel, forceps, spatula,)
- Ketamine / Xylazine cocktail
- Isoflurane
- 1mL Disposable syringe + 25g PrecisionGlide needle
- 60mL syringe
- 10mL Syringe
- 1ft surgical tubing x 2
- Filed 25g needle tip (files using sandpaper to flat tip)
- Glass vials w labeling tape
- Proper PFA / Hazardous material disposal containers: solid & liquid PFA Waste
- Biohazard / mouse carcass bags
For Vibratome slicing:
- Leica VT1000 S vibratome
- Vibratome blade holder and placement knob
- Vibratome slicing blade
- Vibratome hex-screw tightening tool
- Removable stage holder with tightening screw
- Removable stage
- Personna 0.012 HD Heavy Duty Single Edge Razor blades
- Hard plastic guillotine brain holder w blade slits
- Scalpel + blade
- Super glue
- spatula
- 6 well plate
- Glass vials + labelling tape
- Loew-Cornell size 0-3 round brush
- Zeiss Axioskop 2 Plus
For Antibody staining & mounting:
- Triton X-100 Normal
- Normal Donkey Serum (NDS)
- Primary Antibody (refer to attached table)
- Secondary Antibody (refer to attached table)
- Plastic transfer pipettes
- Foil wrap
- Glass petri dish x2
- Loew-Cornell size 0-3 round brush
- Glass vial (1 / required antibody cocktail)
- ProLong Diamond Antifade Mountant
- FisherScientific Premium Frosted Microscope Slides 3” x 1” x 1mm Glass
- FisherScientific Premium Cover Glass 24 x 50, Thickness: 1
- Heathrow Scientific Slide Box
Solutions:
- 0.2 M Phosphate Buffer (PB):
- PB solution A: 0.2 M Na2HPO4.7H2O (MW: 268.07; 53.614 g/L)
- PB solution B: 0.2 M NaH2PO4.H2O (MW: 137.99; 27.598 g/L)
- 8% formaldehyde in dH2O
Fixative preparation
Required PPE: Safety glasses or visor, lab coat, 2 x gloves.
Note: Carry out all work in the fume hood. Use dedicated glassware and thermometer. Maintain a separate bottle of dH2O so that formaldehyde-contaminated glassware does not come into close proximity with the water purification system.
1. Make up 0.2 M Phosphate Buffer (PB):
- PB solution A: 0.2 M Na2HPO4.7H2O (MW: 268.07; 53.614 g/L)
- PB solution B: 0.2 M NaH2PO4.H2O (MW: 137.99; 27.598 g/L)
- Mix A & B at ~5:1. Add B gradually to A until the pH 7.4 reached. 0.2 M PB can be stored at 4 ◦C for up to 2 weeks.
2. Make up 8% formaldehyde in dH2O:
- In 500 mL flask, heat 200 mL of dH2Oto ~50◦C (do not go above 53 ◦C)
- Add 20g granular paraformaldehyde
- Add 1M NaOH with a glass pipette until solution is clear (~20–25 drops)
- Let solution cool and filter with glass funnel and filter paper
- Make solution up to 250 mL with dH2O in graduated cylinder
3. Combine 250 mL of 8% formaldehyde and 250mL of 0.2M PB pH7.4 to make 500mL of 4% formaldehyde in 0.1 M PB pH 7.4. Check pH with pH paper. Store at 4 ◦C in fridge designated for PFA storage, use within 3 days of making (ideally same day).
Mounting / Storage details
- Proper slide labelling format is mouse ID – hemisphere series primary antibody secondary antibody genotype initials – date
- Insert new slide data and location into slide census spreadsheet
Perfusion fixation
Perfusion fixation
While wearing proper PPE, set up surgical area in fume hood
Once a suitable period post-surgery has occurred (3+ weeks) the animal can be perfused
Set up surgical area
Load ketamine-xylazine cocktail into injection syringe (0.2mL/mouse)
In physiological fume hood (separate from PFA-exposed hood) load isoflurane into holding chamber
Place mouse in isoflurane holding chamber until mouse breathing has slowed and running has stopped
Inject 0.2mL ketamine/xylazine IP and place animal back into original cage
Monitor breathing and foot-pinch twitch reflex
Once mouse reflex has stopped (should still be breathing) quickly move mouse to the surgical area
Restrain mouse using PrecisionGlide needles to hold down limbs
Cut mouse skin from to reveal fascia layer, do not yet make incision to reveal pleural space
Using fine surgical scissors, cut fascia to reveal organ space
Cut diaphragm and up sides of rib cage to reveal heart
Using resistance clippers, cut right atrium of the heart
Grip heart and insert cold PBS flat needle into left ventricle from the apex of the heart, begin to perfuse 10mL of PBS by hand at ~2mL/min
Begin flow of PFA at 120mL/hr, switch PBS needle for PFA needle using the same hole formed from the initial insertion
Set timer for 10min, when expires reduce flow to 100mL/hr for 15min
Set timer for 15 min, when expires reduce flow to 90mL/hr until 50mL is reached, 25min
Remove needle, turn mouse over (should be rigid due to PFA perfusion) and use thick scissors to sever head
Using fine surgical scissors, remove scalp to reveal skull
Use resistance clippers to gently cut skull without damaging brain, make incisions on skull at most rostral section of brain to enable skull to be peeled from brain
Use spatula to delicately remove brain and place into glass vial labelled with mouse ID, 4% PFA, initials and date
Refrigerate brain at 4°C for 24 hours
Brain Slicing - Vibratome Operation (24 hours post-perfusion)
Brain Slicing - Vibratome Operation (24 hours post-perfusion)
Set up work-station by gathering 6-well plate with PBS, a petri dish, and 6/12 glass vials (depending on if collecting both hemispheres)
Fill all of these with PBS, label the glass vials with the mouse ID, hemisphere genotype, initials, and date
Remove vibratome blade from manufacturer packaging and wash with ethanol followed by distilled water to remove protective oils
Use hex-tool to tighten blade to the blade-holder so that it is straight and extends several millimeters beyond the black blade-guards
Do not attach blade to vibratome at this time
Plug in vibratome and turn on, check settings: Slice thickness is 70um, slice frequency is set at 9, slicing speed should be between “4” and “5”
Retrieve brain from 4°C Fridge, wash in PBS x 3 and dispose of waste in Liquid PFA waste disposal unit
Place brain into holding chamber and using a brush, gently orient the brain to be equally distributed along the sagittal axis
For sagittal slicing, take one Personna 0.012 HD Heavy Duty Single Edge Razor blade and insert into the sagittal guide slits, push down through brain while maintaining even force between sides of the blade, this will cut the brain into two halves
Take one hemisphere and place into a petri dish filled with PBS, put the other half back into glass vial of PBS and store at 4°C until ready to slice
Equip the scalpel with its blade and prepare to use
Using spatula, pick up the hemisphere by the bisected plane on the flat edge of the spatula
Using filter paper, dry the bisected plane, absorbing residual PBS
Paint a thin line of super glue onto the vibratome stage, then quickly use the flat side of the scalpel blade to push the onto the stage so that the bisected plane comes into full contact with the glue
Place stage in stage holder and fill with PBS
Attach vibratome blade to vibratome
Raise the stage so that the blade is several millimeters above the slice
Using V-Max, set front and back of continuous cutting using the limit-set button, should be set a few millimeters in front of and behind the most forward and back parts of the brain
Begin slicing, placing the first brain slice in well 1, and the following in well 2, going in 6 slice groups (slice 7 will be in well 1) to generate a 1/6 series of the brain per vial
Once complete, take slices from ONE WELL that should contain your ROI and observe for viral expression using the epifluorescent microscope (if applicable)
Return slices to their original well
Transfer slices
Primary incubation
Primary incubation
Place each series in a glass vial and rinse sections with PBS 3 times
Add 1 mL of PBS-T with 2% Normal Donkey Serum (20 µL/mL) to each series and swirl briefly
Optional blocking step: leave slices in PBS-T and 2% Normal Donkey Serum at room temperature for 45–60 min
Add primary antibody to each series (see table 2)
Shake gently for 48–72 h at 4 ◦C (sections should barely revolve around the vial)
Secondary incubation
Secondary incubation
Rinse sections with PBS 3 times before starting secondary reactions
Create necessary “Secondary Antibody Cocktail” consisting of 1 mL of PBS-T with 2% Normal Donkey Serum (20 µL/mL) and corresponding secondary antibody (refer to suggested antibody concentration). Volume of cocktail should be +1 to all reagents to avoid lack of volume due to pipette error (meaning 12 vials = 13mL PBS-T, 20uL NDS x 13, 4uL 2° x 13)
Protect from light for all remaining steps.
Shake gently for 90 min at room temperature (sections should barely revolve around the vial)
Rinse sections with PBS 3 times before mounting
Using a glass petri dish, gently remove brain slices from 1 vial at a time and mount onto “name of glass slide”
Mounting slices and Labelling
Mounting slices and Labelling
Mount sections serially on slides with Prolong Diamond Anti-fade mounting media; protect slides from light and keep at 4 ◦C after 24 h drying at room temperature
Proper slide labelling format is
mouse ID – hemisphere
series
primary antibody
secondary antibody
genotype
initials – date
Insert new slide data and location into slide census spreadsheet