Immunofluorescence of Paraffin Embedded Sections 

Diana Pastrana, Buck Lab, LCO

-       Based on two Protocols: 

1. Robertson, D., Savage, K., Reis-Filho, J. S., Isacke, C. M., March 2008. Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (ffpe) tissue. BMC Cell Biology9, 13+.
2. Shuda, M., Arora, R., Kwun, H.J., Feng, H., Sarid, R., Fernandez-Figueras, M.T., Tolstov, Y., Gjoerup, O., Mansukhani, M.M., Swerdlow, S.H., Chaudhary, P.M., Kirkwood, J.M., Nalesnik, M.A., Kant, J.A., Weiss, L.M., Moore, P.S., Chang, Y. 2009. Human Merkel Cell Polyomavirus Infection I. MCV T antigen expression in Merkel Cell carcinoma, lymphoid tissues and lymphoid tumors.  Int. J. Cancer

MATERIALS:

-       10% Neutral buffered Formalin (Fisher #23-032-060 orProtocol#032-060) or 2% Paraformaldehyde (Electron microscopy #RT 15710)

-       Optional: PBS/200 mM Glycine, 0.02% Sodium azide (Make the solution and filter sterilize, it has good shelf-life)

-       Paraffin Embedding service (Histoserve) ask for sialanated slides (Cat# ST002C includes embedding and sectioning they charge 5.50/slide)

-       Rotary Microtome for cutting sections (or have histoserve do it)

-       Water Bath at 95°C

-       Staining Trays (choose between one [100ml capacity 10-12 slides] ortwo [200ml capacity 20 slides])

1. Simport Plastics Easy Dip (Simport #M900-12AS use -12b,g, p, w, y for blue, green, pink, white or yellow) and staining rack for 12 slides (Fisher # 22-038-495 for the assorted)
2. Wheaton Glass 20 slide staining dish with removable rack (Fisher #08-812 for complete set Wheaton #900200or individually #08-812-1A for Dish and cover Wheaton #900203, #08-812-1B for slide Rack Wheaton #900204, #08-812-1C for Handle Wheaton #900205)

-       Xylene (FUME HOOD is necessary!!)

-       Ethanol

-       Target Retrieval solution (Dako # S1700 Ready-to-use or #S199 10x concentrate)

-       Protein Block (Dako# x0909) or(PBS, 1%BSA [bovine serum albumin], 2% FBS [fetalbovine serum] filter sterilized) 

-       Optional: ImmEdge Hydrophobic Barrier Pen (Vector laboratories #H-4000 or Fisher NC9545623)

-       Plastic Coverslips (Rinzl Coverslips from Electron microscopy sciences #72261-23 or Fisher #S17525A)

-      Glass Coverslip, uncoated #1 (0.13-.17mm thick, squares 25mm) Fisher#15-542C

-       Optional: PBS/0.1% Brij 58 (Make a 1% solution of Brij in PBS by warming up to 37°C, then use this to make the solution, also filter sterilize the solution and keep both stock brij and solution for about 2 months)

-       Optional: Humidified chamber (a container filled with damp paper towels in the bottom and sealed with parafilm)

-       Alexa 488 or 594 secondary antibodies (i.e. invitrogen goat anti-mouse Alexa 488 #A11029)

-       Prolong Gold Antifade with DAPI (Molecular Probes Cat# 36931)

METHOD:

-       Fix tissue in the 10% Neutral buffered Formalin (or 2% Paraformaldehyde diluted in PBS).  If samples are trypsinized cells or monolayers do 20 min room temperature. If the samples are whole tissues cut into appropriate size and place in Formalin Overnight at room temperature. (For trypsinized cells send 5 million cells) Try to do end over end mixing of cells/tissues

-       Rinse in water (alternatively could wash in PBS/200 mM Glycine, 0.02% Sodium azide [Make the solution and filter sterilize, it has good shelf-life rinse] 3x over 5-10 min) or in PBS

-       Histoserve asks to send samples in 70% EtOH, but that just seemed to dissolve the cells, it is probably better to do PBS/glycine followed by one PBS rinse

-       Send to Histoserv Inc for paraffin embedding and sectioning or section in-house

Deparaffinization and antigen retrieval

-       If slides are not silanated or you are unsure, bake the slides for 1 hour at 60°C.  You can use a heat block with the heating inserts inverted (holes down).  Make sure you cover the slides so dust does not deposit on them

-       Prepare solutions and place them into staining jars

-       Warm up the water bath to 95°C

-       Place slides into holder that will be dunked in staining dishes and perform the following steps:

A)   RinseTWICEin 100% xylene for 5 min. with agitation every 30 secs.  (Change reagent between the two rinses, to discard xylene allow it to evaporate in the fume hood)

B)   100% Ethanol 20 seconds to 1 minute

C)   90% Ethanol 20 seconds to 1 minute

D)   70% Ethanol 20 seconds to 1 minute

E)    WaterTWICE 20 seconds (change water between rinses) to 1 minute

F)    Place in jar containing Target Retrieval solution (warmed up to 95°C) in a water bath for 30min

G)   Remove entire jar from water bath and cool at room temperature for 20 min.

H)   Rinse 3x in water (Robertson recommends 5 min running water, Dako recommends 3 rinses in buffer)

Staining

-       If you plan to use it, rinse water around tissue and make a circle with ImmEdge pen and shake off excess water.  Add PBS

-       If the pen is not used rinse once in PBS

-       FOR PARAFFIN sections block as follows: use Dako protein block (or PBS/1% BSA/2%FBS or PBS/0.1% Brij58/2%BSA) for 10 min at room temperature with GENTLE rocking.  If not using the pen all reagents should be added as follows: place 60µl of solution onto the tissue and place a plastic coverslip on the slide, if a long time is needed for the stain place in a humidified chamber 

-       For Cryosections Block as follows: Block with 10% serum of the species used for secondary, usually from Jackson Immuno-research.  For example normal goat serum (# 005-000-121) comes as freeze dried powder that gets reconstituted in water.  After this, the stock is 60 mg/ml, so the final blocking concentration is 6 mg/ml in PBS/0.1% Brij 58 (Make a 1% solution of Brij in PBS by warming up to 37°C, then use this to make the solution, also filter sterilize the solution and keep both stock brij and solution for about 2 months).  For normal slides calculate 200 µl of block per slide and you can block at the bench for 1 hour WITHOUT a coverslip

-        

-       Add primary antibody diluted in PBS/0.1% Brij for 1-2 hours at 37°C with plastic coverslip.   Alternates are: room temperature rocking (or 4°C overnight without rocking). Alternatively can use block or plain PBS for antibody dilution

-       Gently tilt slides and allow the coverslip to migrate to the bottom.  Grab it with the hand or tweezers (can use other side for secondary antibody after blotting).  

-       With a 10ml pipette gently rinse the coverslip with PBS/Brij. Then place slides in a suitable container (pipette tip box) containing PBS/Brij and rock gently once for 15min. 

-       Add Secondary antibody (i.e. invitrogen goat anti-mouse Alexa 488 #A11029 use at 1:1K) for 30-60min room temperature rocking with plastic coverslip

-       Gently tilt slides and allow the coverslip to migrate to the bottom.  Grab it with the hand or tweezers (can use other side for secondary antibody after blotting).  

-       With a 10ml pipette gently rinse the coverslip with PBS/Brij. Then place slides in a suitable container (pipette tip box) containing PBS/Brij and rock gently once for 15min. 

-       Get rid of excess liquid by blotting edge of slides

-       Add Prolong gold mounting medium and place glass coverslips on top.  Cure overnight, flat and in the dark.  Afterwards store at 4°C or -20°C.

-       Visualize in confocal microscope.  

NOTE: If you absolutely have to use an antibody raised in the same species as the tissue you are staining do the following

1)   Block as normal with normal serum same as secondary antibody (i.e. if staining with Mouse monoclonal antibody and using a Goat-anti mouse Alexa conjugate, block with normal goat serum)

2)   Block with Affinipure Fab fragment unconjugated antibody of the same species as secondary, so for our example use Goat anti-mouse Fab (0.1 -0.3 mg/ml)

3)   Wash it away

4)   Stain with primary as usual and wash as usual

5)   For secondary: stain with Affinipure Fab antiserum  (Fab goat anti-mouse Alexa fluor 488 conjugated).