Immunohistochemistry

Immunostaining of mouse liver frozen sections

Tissue Preparation [I] for mouse embryos, pups and mice aged up to P21 (pre-weaning)

1. Dissect the embryos or tissues, quickly rinse with cold PBS and fix overnight with fresh 4% PFA in PBS (at least 10 volumes), at 4^oC.
2. Transfer the tissues to 30% sucrose in PBS (at least 10 volumes) and rock overnight at 4^oC.
3. Cover the tissues with Tissue-Tek (OCT) and freeze on dry ice. 
4. Section the specimens with a cryostat (10 µM). Sections can be stored at –20^oC or processed immediately as indicated below.

Tissue Preparation [II] for mice aged >P21

1. Mice P21 and older should undergo intracardial perfusion with 4% PFA in PBS prior to harvesting the organs.
2. Remove the organs and place in a plastic tube with 10 volumes of ice-cold 4% PFA.
3. Post-fix tissue at 4^oC, for 5 hours on a rocker.
4. Remove the PFA and briefly wash once with ice cold PBS.
5. Add 10 volumes of ice cold 30% sucrose in PBS and incubate overnight in the cold room on a rocker. Check that there is no adhering fat, as this will prevent the tissue from submerging in the sucrose.
6. Ensure that the tissue has sunk to the bottom of the sucrose solution before proceeding to embedding as described below.
7. Cover the tissues with Tissue-Tek (OCT) and freeze on dry ice. 
8. Section the specimens with a cryostat (10 µM). Sections can be stored at –20^oC or processed immediately as indicated below.

Immunostaining

Day 1

1. Allow slides to warm to RT for 5-10 minutes.
2. Outline sections with hydrophobic pen.
3. Wash with TBS-T (TBS + 0.05% Tween 20) for 10 min on a rocker.
4. Place slides in a humidified chamber and block for 30 min @ RT with blocking solution (enough to cover the section; do not overflow).

Blocking solution:

a) Maleate buffer: 100 mM maleic acid, 150 mM NaCl, pH 7.5 adjusted with NaOH. 

b) Blocking reagent (Sigma cat No 11096176001). Dissolve the blocking reagent in maleate buffer to a final concentration of 10%, store it @ -20^oC as 10 ml aliquots. 

c) Horse serum (heat inactivated, sterile, SIGMA cat No H1138).

d) Mix: 3 mL Maleate buffer + 1 mL Horse serum + 1 mL 10% Blocking reagent. Use within 1 week and keep at 4^oC.

1. Remove blocking solution and add primary antibody diluted in blocking solution to the sections (30-50 µl is sufficient for a 1cm² section).
2. Incubate O/N in a humidified chamber at RT.

Day 2

1. Remove the primary antibody by aspiration and wash 3X with TBS-T, 10 min ea. and rocking.
2. Incubate with fluorescent-labeled secondary antibody diluted in blocking solution, for 2-3 h @ RT.
3. Remove antibody and wash 3X with PBS, 5 min ea.  and rocking.  
4. Incubate the slides with DAPI (diluted 1:1,000 in PBS) for 5 min @ RT
5. Wash 1X with PBS, 5 min.
6. Wash 2X quickly in dH2O (“Dip and remove”).
7. Dry the sections for ~20 min, mount with ProLong^TM Gold (ThermoFisher cat No P10144) and coverslip.

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