200 g paraformaldehyde

1 ml 10N NaOH

up to 1 liter with Q, heat to 65° to dissolve, aliquote and store at -20°

Mix 100 ml 20% Paraformaldehyde with 50 ml 10X PBS and bring up to 500 ml with Q

filter, and store at 4° for up to 2 weeks

• Dissect and fix the tissue in 4% paraformaldehyde on ice for 5-10 minutes.

• Wash twice in PBS for 5 minutes each.

• Dehydrate the tissue using an alcohol series:

(50% MeOH:PBT) x 2

(100% MeOH) x 2

(100% EtOH) x 2

• Transfer to glass scintilation vials, pour in xylene (in hood) and incubate for 10 minutes at room temperature.

• Remove xylene and add a 1:1 mixture of parafin:xylene and incubate in 56-60° oven under vacuum for 30 minutes.

• Repeat the previous step with 100% parafin.

• Transfer the tissue to the metal mold filled with hot parafin and allow to cool at room temperature.

• Store at 4° for several months.

• Cut 5-6 micron sections in multichamber with 10% EtOH, transfer to silinized (Protocol S.6) slides and dry overnight at 42°C.

• Proceed with immunohistochemical detection or store at 4° dessicated for several months.

• Dewax for 10 minutes in Xylene in the hood, repeat.

• Rehydrate through an alcohol series starting with two washes in 100% EtOH followed by 95%, 90%, 80%, 70%, 50%, 30% and 2 washes in PBS for 5 minutes each.

• For in situ hybridization proceed with proteinase K digestion for immunohistochemistry block for 30 minutes as usual.