Bitan:frozen section IHC

Nov 2013 Aida Attar

IHC protocol for frozen slides (3/2/12) From Ed Teng / Fusheng Yang Done by Tanya Liu and Ryan Guglietta and Aida Attar

DAY 1: Pretreatment of slides

For brains that have NOT been fixed (paraformaldehyde was NOT used in perfusion) Fix the tissue sections with a suitable fixative. One of the commonly used fixation methods for frozen tissue sections is to immerse the slides in pre-cooled acetone (-20°C) for 10 min. Pour off the fixative and allow acetone to evaporate from the tissue sections for > 20 min at room temperature. 1. When required, leave to warm at room temperature for 5 min. 2. Pre cool the fixative (acetone, methanol or ethanol) at -20°C for 30 min. (Abcam recommends starting with acetone) 3. Fix with the pre cooled fixative for 5-10 min, at room temperature. 4. Rinse 3-4 X in PBS. 5. Continue with the immunohistochemical staining protocol. The absence of a formaldehyde based fixative eliminates the need for an antigen retrieval step. However, if frozen tissue or cytological specimens have been fixed in formalin, antigen retrieval can be attempted, although the friable nature of the specimens, in particular brain tissue, may compromise the success. The following reference describes a protocol in which slides are treated with 3-aminopropyltriethoxysilane (APES) before placing sections on the slides. This treatment improved adhesion, allowing heat mediated antigen retrieval with minimal damage to the tissue morphology: Warembourg M, Leroy D., Microwave pre treatment of sections to improve the immunocytochemical detection of progesterone receptors in the guinea pig hypothalamus. J Neurosci Methods. 2000 Dec 15;104(1):27-34. A more thorough discussion of antigen retrieval applied to frozen tissue sections is found in the following reference: Yamashita S, Okada Y. Application of heat induced antigen retrieval to aldehyde fixed fresh frozen sections. J Histochem Cytochem. 2005 Nov;53(11):1421-32.

-dry for 10 minutes at room temp

-TBS 1x 5-10min (PBS ok)

-TBST (0.01% tritonX-100) 1x 10min

Pap pen -methanol + 0.3% H2O2 for 10min for 200mL container: 2mL of 30% H2O2 in 198 mL methanol OK to do .3% H2O2 in PBS

Rinse in PBS/TBS

antigen retrieval by steaming for an hour if fixed at all or as follows (cryosectioned brain processing protocol: all steps should be done on shaker place 1st day: Formalin (10% solution) fixing 8-16 hrs (O/N); 2nd day: wash in PBS 1 hr x 3 on, 10% sucrose in PBS O/N 3rd day: 20% Succrose in PBS 6-8 hrs (or O/N) until brain sink to bottom of bottle Snap freeze in chilled 2-methybututane around -(70 C) and store in -70 C freezer (about 18 sec))

-block for 45 min @ 37 C (optionall??) 5% NGS in 3%BSA/TBST

-TBS 1x 5min

-primary antibody for 1 hr @37 C, then O/N @ 4 C DAE 1:200 in 3%BSA/TBST

DAY 2

-TBS 3x 5min In her email, she said it’s suppose to be 1.5% NGS but in her notes, she did 15 uL NGS in 200 uL which is actually 7.5% NGS

-secondary antibody for 45min @ 37 C anti-rabbit 1:1000 + 1.5% NGS in 3%BSA/TBST In her email, she said it’s suppose to be 1.5% NGS but in her notes, she did 15 uL NGS in 200 uL which is actually 7.5% NGS

-ABC: 1% A, 1% B in TBS for 45min @ 37 C remember to make 30 mins prior to use (leave on ice or in fridge)

-TBS 3x 5min

-DAB?? 1) She uses 1:10 DAB in buffer. We do 1 drop of DAB for every 1mL of DAB diluent (which, according to the kit’s protocol sheet, is about 1:30) She puts the DAB in “DAB H2O2 Buffer”—what is that? We put our DAB into DAB diluent (given with the kit)

-ddH2O 1x 5min

-70% EtOH 5min -96% EtOH 5min -100% EtOH 5min

-CtriSolv 2x 10min

-coverslip with permount and dry in hood overnight

-clean slides with CitriSolv