Day 1
Note: take care to keep sections hydrated at all times. Drying out will lead to non-specific staining.
- De-paraffin and rehydrate slides
- Incubate 5’ each in xylenes (x2), then 3’ each in 100% EtOH (x2), 95% EtOH (x2) and 70% EtOH (x2)
- Put slide rack in PBS while preparing the next step
- Antigen retrieval in 10 mM citrate buffer pH 6.0i
- Pre-heat buffer for at least 30 minutes before adding slides
- Incubate slides in buffer at 95oC-100oC for 25 minutes
- Let slides cool in AR buffer at RT for 30’
- Put slides in PBS while preparing the next step
- Our citrate buffer is made with citric acid (others are made with sodium citrate). Note you may also want to try Tris-EDTA pH 9.0 (10 mM Tris base + 1 mM EDTA and tween 0.05%) if you get poor staining with Citrate, this buffer may give higher background for some antigens but you can also often use the antibody at a more dilute concentration (and it has worked really well for some antibodies that did not work in citrate, such as GATA3)
- Block endogenous peroxidases with H2O2
- Make up 3% H2O2 fresh (180 ml PBS + 20 ml 30% H2O2)
- Incubate for 10’
- Put slides in PBS while preparing the next step
- Block in PBS + 1% BSA and 2% serum from the host of the secondary antibody (horse for anti-mouse, goat for anti-rabbit; use 20 ml per ml)
- Segregate tissue sections from each other with a Pap Pen if necessary
- Add 100-200 ml of block to each tissue spot (depending on size and use of pap pen)
- Incubate at RT for 1 hour in a humidified chamber
- Bind tissues with primary antibodies
- Make up primary antibodies in PBS + 1% BSA at the appropriate dilutions, plan on 150 ml per section if using the pap pen. If not using the pap pen or sections are large, scale up (this goes for all subsequent steps mentioning 150 µl)
- Incubate overnight at 4C in a humidified chamber, if desired, use probe clips for sections with larger area and/or only one section per slide
Day 2
- Wash slides in PBS 3 x 5’, can let slides soak in last PBS wash for 1 hour or so if needed (note, these washes can be sped up if necessary)
- Bind tissues with secondary antibody
- Make up secondary antibody at 1:200 in PBS (we use the Vector Labs biotinylated secondary antibodies, cat# BA-2000 for anti-mouse and cat# BA-1000 for anti-rabbit)
- Incubate at RT 30’ in a humidified chamber
- Prepare ABC reagent-Make at least 30’ before use
- Add 2 drops reagent A and B to 5 ml PBS (we use the Vector Labs ABC elite standard kit, cat# PK-6100)
- Wash slides 2 x 5’ in PBS
- Add 150 ml ABC reagent to sections and incubate at RT 30’
- Wash slides 2 x 5’ in PBS, keep slides in PBS until development
- Prepare Nova Red* stain immediately before use and re-make if it sits longer than 10-15’ min; Alternatively, use ImpactDAB** stain and follow instructions with the kit (can be prepared ahead, steps b and c will be the same).
- To 5 ml tap water, add 3 drops reagent 1, 2 drops reagent 2, 2 drops reagent 3 and 2 drops hydrogen peroxide
- Add to sections, incubate until stain develops to desired darkness
- Cover sections not developed at the same time with water to prevent dry-out; place finished slides in water
*Both NovaRed from Vector labs (cat# SK-4800) and ImpactDAB substrate (cat# SK-4105) work well-DAB is more of a brownish stain and NovaRed is obviously red. Some uses have found that the NovaRed stain leaches off the slides if they are left too long in 70% EtOH (see below) so we have converted to mostly using DAB.
- Counterstain briefly with 1:5 hematoxylin* in water (about 10-15 seconds)
- Dip into water until clear
*this will vary with your formulation of hematoxylin
- Dip sides in 2% glacial acetic acid made up in dH20, dip in water, Dip in 0.1% ammonium hydroxide made up in dH20, dip with water
- Dehydrate to xylenes through graded ethanols (3’ in 70% ethanol x2, 1’ in 95% ethanol x 2, 1’ in 100% ethanol x 2, 5’ in xylenes x2), note that long incubations in 70% ethanol removes some NovaRed staining, which can be good or bad, depending on your needs
- Mount with permount and coverslip, allow to dry in the hood
