Lacz Stain for Slide-Mounted Fixed Tissue

LacZ Stain for Slide-Mounted Fixed Tissue (adapted from Nirao Shah, UCSF, Spring 2013)

Tissue preparation: 1. Perfuse mice for 5 min each with 1xPBS containing 10 U/ml heparin, followed by 4% PFA/1xPBS. Harvest brain. 2. Immersion fix brain in 4% PFA/1xPBS for 3 hr at 4º C. Be careful not to overfix tissue or enzymatic activity of -galactosidase will weaken. 3. Transfer to 30% sucrose/1xPBS at 4º C. 4. When tissue has sunk (24 – 48 hr), section on freezing sliding microtome at a thickness between 20 and 60 μm (35 μm for standard tissue, 45 μm for gelatin embedded brains). 5. Keep sections at -20º C in antifreeze solution. 6. Mount sections onto slides and let dry at room temperature for 24 – 48 hr.

LacZ stain: 1. Wash slides in Solution A for 5 min at room temperature. All steps should be performed in Coplin jars or other glass histology dishware. 2. Wash slides again in Solution A for 15 min at room temperature. 3. Incubate in pre-warmed Solution B for 10 min at 37º C. 4. Incubate in pre-warmed Solution C at 37º C in the dark. The length of development will depend on the origin of your tissue (strength of driver/responder line, transgene copy number, etc.) and can require several days in some cases. If the stain requires more than 24 hours to develop, replace Solution C every 24 hr. We have found that 30 min incubation is sufficient to develop signal from a relatively strong tTA-driver/tetO-lacZ responder pair (Neuropsin-tTA/tetO-nls-lacZ; Yetman, Lillehaug, et al. 2014). 5. Wash slides 3x for 5 min each in stop solution at room temperature. 6. Incubate slides in 4% PFA/1xPBS for 1 hr at 4º C to fix tissue. 7. Wash slides 3x in 1xPBS. 8. Dehydrate tissue through graded ethanol series into xylene (i.e., 2x 70% EtOH, 4x 95% EtOH, 4x 100% EtOH, 4x sylene). Cover slip with Permount.

Nuclear Fast Red counterstain (optional):

9. After dehydrating through the series of increasingly hydrophobic solutions, rehydrate in reverse order back into water. NOTE: Dehydration and rehydration may not be necessary if you plan to counterstain in NFR. We have always performed this protocol with these steps included, but are not sure they are needed. 10. Incubate at room temperature for 1-2 min in a 1-to-3 dilution of nuclear fast red stock solution (0.1% nuclear fast red Kernechtrot (Fluka, 60700) with 5% aluminum sulfate hydrate (Mallinckrodt, 3208)) until the sections attain a light pink color. Alternate between water and staining solution to gradually get the desired staining intensity. It can be helpful to check the color after 30 sec in stain and a quick rinse before returning to stain for the final 1 - 1.5 min. 11. Dehydrate as in step 8. Coverslip with Permount. Solutions:

Solution A: 1x PBS

2 mM MgCl2

Solution B:

0.1 M NaPO4 2 mM MgCl2 0.2% NP-40/Igepal 0.1% Sodium Deoxycholate

Solution C (10 ml): 9.35 ml of pre-warmed Solution B 0.5 ml of 200 mM ferro/ferri cyanide stock 0.15 ml of X-gal, 40 mg/ml in dimethylformamide (5 PRIME, #2500040) (store X-gal stock solution in dark at -20º C, discard if precipitate appears)

10x Hepes Buffered Saline (HBS) Stock: 29.75 g HEPES 41 g NaCl

3.7 ml of 1 M Na2HPO4 ~1.6 ml of 10 N NaOH (to bring final pH of HBS solution to 7.0)

dH2O to 500 ml

Stop Solution: 1x HBS, pH 7.0 0.1% Triton X-100 1 mM EDTA

200 mM ferro/ferri cyanide stock 200 mM Potassium hexacyanoferrate (III) (Sigma, #455946) 200 mM Potassium hexacyanoferrate (II) trihydrate (Sigma, #455989)

to volume with dH2O (store in dark at room temperature)

1 M Phosphate Buffer, pH 7.4

77.4 ml of 1 M Na2HPO4 22.6 ml of 1 M NaH2PO4

Antifreeze Solution (for free-floating sections) 250 ml Glycerol 300 ml Ethylene Glycol 500 ml 0.1 M Phosphate Buffer, pH 7.4