Preparation day 1	1× PBT, nuclease free: 1× PBS + 0.1% Tween 20
4% PFA in PBS, pH 7
Sterile nuclease free 1 × PBS
Perfusion materials
MeOH solutions in PBT (50%, 75%, 100%)
Heat water bath or incubator to 40 °C with gentle agitator/rocker if available
Sample collection/fixation	Transcardially perfuse mice with 4% PFA in PBS
Remove and microdissect tissue of interest in 0.4% PFA being sure to remove all extraneous tissue
Immersion-fix tissue in 4% PFA/PBS for 1 h (ear) or 2 h (thick brain sections) at room temperature (RT) on rocker
Transition	Wash tissue 3 × 10 min in PBT
Pretreatment/dehydration	Dehydrate tissue through a graded MeOH/PBT series: 50%, 75% for at least 5 min each at RT on rocker
Transfer tissue to 100% MeOH for at least 5 min.
Note: Can store tissue in 100% MeOH at − 20 °C if needed
Rehydrate	Reverse MeOH/PBT series as above
Transition	Wash tissue 3 × 5 min in PBT at RT on rocker
Protease digestion	Protease III 1× solution (see Table 1 for incubation times) at RT on rocker
Stop digestion	Wash tissue 3 × 5 min in PBT at RT on rocker. Start to warm probes (see below)
Prepare probe(s)	Pre-warm probes to 40°C (10min) and then cool down to RT (~ 10min). This ensures there is no precipitation
Invert probe solutions to mix. Briefly spin the C2 and/or C3 probes to collect liquid at the bottom of tubes Mix probes: channel-1 probe (C1; 1×), channel-2 (C2; 50×), channel-3 (C3; 50×). To make a multiplex mixture at 1× concentration, mix C2 and/or C3 probes 1:50 with C1 probe
Hybridization	Incubate tissue in probe mix at 40°C with light rocking. See suggested hybridization times in Table 1
Preparation day 2	0.2× SSC: filute 20× SSC with sterile nuclease-free dH2O
Heat water bath or incubator to 40°C Equilibrate FL-AMP1–4 reagents (1×) to RT and invert to mix before use If using, remove Prolong Diamond Mountant from −20°C and place at 4°C for 24h or RT for ~ 6h
Probe removal	Recover the probe solution in a new tube; these can be reused.
	Label and store at 4°C for up to 6months
Wash	Wash the tissue 3 × 15min with 0.2× SSC at RT on rocker
Postfixation	Fix tissue again with 4% PFA in PBS at RT for 10min
Wash	Wash the tissue 3 × 5min with 0.2× SSC at RT on rocker
Preamplifier hybridization	Remove 0.2× SSC and replace it with Amp1 solution (1×)
Incubate at 40°C for 35min with gentle rocking (best practice) or invert tube regularly
Wash	Wash the tissue 3 × 15min with 0.2× SSC at RT on rocker.
Signal enhancement	Remove 0.2× SSC and replace it with Amp2 solution (1×)
Incubate at 40°C for 20min with gentle rocking (best practice) or invert tube regularly
Wash	Wash the tissue 3 × 15min with 0.2× SSC at RT on rocker
Amplifier hybridization	Remove 0.2× SSC and replace it with Amp3 solution (1×)
Incubate at 40°C for 35min with gentle rocking (best practice) or invert tube regularly
Wash	Wash the tissue 3 × 15min with 0.2× SSC at RT on rocker
Label probe hybridization	Remove 0.2× SSC and replace it with Amp4 solution (1×). [Use desired reagent, Alt A, B, or C]. LIGHT SENSITIVE! Protect from light!
Remember the combos: ALTA=C1(488), C2(550), C3(647); ALTB=C1(550), C2(488), C3(647); ALTC=C1(550), C2(647), C3(488) Incubate at 40°C for 20min with gentle rocking (or invert tube occasionally)
Wash	Wash the tissue 3 × 5min with 0.2× SSC at RT on rocker. Note: Temporary storage (1day) in 0.2× SSC is possible before mounting
IF/counterstain	Remove 0.2× SSC and add fluorescent nuclear staining solution if desired and/or proceed with immunofluorescence (IF) according to protocol
Wash	Wash the tissue 3 × 5min with 1 × PBS at RT on rocker
Mount/view	Warm the bottle of ProLong® Diamond Antifade Mountant to room temperature before use
	Remove excess moisture from the slide before ProLong® Diamond Antifade Mountant is added. Flat mount samples
	Cure the sample: Place the mounted sample/slide on a flat, dry surface for 24h at room temperature protected from light before viewing