Protocol 3: Immunofluorescence on Frozen Sections

1 Protocol 3: Immunofluorescence on Frozen Sections Brigitte Arduini, version 1, 2015-Mar-23 Modified from Manipulating the Mouse Embryo 3rd edition (2003). Nagy, Gertsenstein, Vintersten and Behringer, ed. Materials Phosphate Buffered Saline (PBS, 1X) Paraformaldehyde (PFA, 4%) see Support Protocol 1 Microscope slides, pre-coated Optimal Cutting Temperature (OCT) Compound 30% sucrose in PBS Cryotome blades (disposable or permanent depending on cryostat) Reagent scoop or slotted spoon Aluminum Foil Conical tube or screw-cap vial Pencil Slide box Humidity box (covered tray with dowels fixed to the bottom) Staining jars/slide holders Cover glass no. TBST solution: 10mM Tris-HCl ( ) + 150mM NaCl + Twe e n 20 50% glycerol solution (diluted in 1X PBS) 4',6-Diamidino-2-Phenylindole ( DA PI, Li f e Te chnol ogi es #D1306) Procedure: Fixation 1. Collect tissue in PBS on ice. 2. Wash 1 2 times in PBS and transfer to a glass vial on ice.

2 3. Cover with 4% PFA and incubate at 4oC for 30 minutes to several hours depending on the size of the tissue. 4. Wash with PBS, 1X f ast f ol l owed by 2 3 times at 4oC over the course of several hours with slow agitation (such as on a nutator). 5. Wash at least twice in PBS for 30 minutes or more. Tissue may be stored at 4oC in PBS before proceeding. Procedure: Preparing Frozen Sections Tissue transfers should be performed delicately, with a slotted spoon or reagent scoop. Use of forceps may damage tissue integrity. 1. transfer tissue to 30% sucrose in a conical tube or screw-cap vial and store at 4oC overnight. Tissue should initially float, and is fully impregnated with sucrose when it sinks1. For larger tissues (such as 1 Sucrose serves as a cryo-pr otec ta nt to pr es er ve c el l ul a r i ntegr i ty of the s a mpl e during freezing. an adult brain), it may be advisable to transfer stepwise first to 20% sucrose overnight, and then to 30% sucrose overnight.

3 2. Using a flat-bottom lab marker, inverted test tube or other suitable object, make a cup-shaped foil mold. The mold should be only slightly larger in diameter and in height than the tissue sample2. 3. Fill the foil mold partially with OCT compound. 4. Remove tissue from sucrose and drain excess solution. Place the tissue sample in the OCT compound, keeping track of tissue orientation. Adjust the orientation with a probe or closed watchmaker forceps if necessary. 5. Add OCT compound if necessary to fully cover the tissue sample. 6. Snap freeze the block in methanol/dry ice, using forceps to hold the edge of the foil mold. Frozen blocks may be stored at -80oC for several days, or immediately sectioned. 7. Mount a disposable blade and place all necessary items in the cryostat: 3-4 kimwipes, brush, etc. Set the cryostat to approximately -28oC and allow temperature to equilibrate. Set the section thickness, typically 12 16 m. 8. Pre-warm slides and label numerically with pencil3.

4 9. Apply a small base of OCT compound to a sectioning pedestal inside the cryostat. Mount the Frozen sample block in this base and press Freeze Object. 10. Make sure that the sectioning arm is moved sufficiently back from the blade that the pedestal and block can be mounted without touching the blade. Mount the pedestal with block in the holder, with the desired orientation. 11. Slowly raise the sectioning arm nearly to the blade. Proceed further advancing the arm as though sectioning, until the blade begins to cut into the OCT block. 12. Wipe away unwanted or damaged Sections with a brush or kimwipe, always moving from the back of the blade to the front. Allow the blade a few moments to return to the proper temperature before resuming sectioning. 13. Sections may be collected singly or in a short string of 2 4 by holding a pre-warmed slide just above the Sections on the blade. Check Sections under a dissecting microscope and keep notes about section location and quality on specific numbered slides for later reference.

5 Return slides to the warm plate to dry. 14. Always clean the blade with brush or kimwipe after collecting Sections and allow temperature to re-equilibrate. 15. Slides should be allowed to dry approximately one hour, and may be stored in a slide box at -80oC for weeks to months before immunostaining. Tips and tricks: If sectioned material begins to shrink immediately, cryostat temperature is too warm. If Sections begin to curl, cryostat temperature is too cold. Distortion of Sections may also result from excess OCT compound surrounding the tissue. 2 Excess OCT compound around the tissue sample will make sectioning more difficult and less consistent. 3 Numbered slides are useful when selecting Sections for staining if, for example, consecutive Sections or specific ti s s ue r egi ons a r e des i r ed. Procedure: Immunostaining Frozen Sections Do not let slides dry out after starting the procedure.

6 1. Warm slides to room temperature for approximately 30 minutes. 2. Cover Sections with ~200 l blocking solution, place in the humidity box and incubate for 20 - 30 minutes at room temperature. 3. Gently flick away the blocking solution and add primary antibodies diluted in blocking solution. Incubate in humidity box at room temperature for 30 minutes to several hours, or overnight at 4oC. 4. Wash slides twice with TBST in a staining jar at room temperature for 10 minutes per wash. 5. Wick away excess TBST with a lab wipe. 6. Add secondary antibody diluted in blocking solution. Incubate at room temperature for 30 minutes to one hour in humidity box. 7. Counter stain with DAPI or nuclear stain of choice diluted in TBST. 8. Wash twice for ten minutes in TBST at room temperature. 9. Mount slides with 50 - 100% glycerol and a no. cover glass. Support Protocol 3: Gelatin Slide Subbing Materials NaOH Solution (1N, 40g/liter) Gelatin solution: 1g gelatin + 1L ddH2O + chromium potassium sulfate Procedure 1.

7 Soak slides in NaOH for at least 15 minutes 2. Rinse slides in ddH2O one time fast, then once for 15 mi nute s 3. Dip slides in gelatin solution 4. Air-dry slides 5. Store slides in slide boxes at room temperature