Postnatal Day 28 mouse lungs were inflation fixed with 4% PFA and incubated overnight at 4oC. After overnight fixation, lungs were washed several times DEPC-treated 1X PBS and dehydrated through a Methanol (MeOH) series to 100% MeOH. Lung tissue was then washed in 3 changes of 100% MeOH for 30 minutes. Once tissue is equilibrated in 100% MeOH, remove air bubbles by squeezing out the air bubbles or by using a vacuum.
Clear in BA:BB 1:2 (1 part benzyl alcohol: 2 parts benzyl benzoate). For a 1.5cm x 1.5 cm x 5mm piece of tissue, it takes ~ 1 hour to clear.
Transfer lungs to fresh BA:BB. Image on a multiphoton microscope with glycerol immersion objectives. Mount tissue in BA:BB in 3D printed sample holders and use a coverglass bottom dish as a lid for the sample. The coverglass bottom dish is filled with glycerol to match the refractive index of the sample. We use either a 10X NA 0.5 WD 5.5mm or a 20X NA 1.0 WD 8.2mm. Image using backscattered SHG with GaAsP NDDs using 840nm excitation and 420/20 filter for SHG, 525/50 for autofluorescence and a 585/50 for pigment (pigment, carbon particles and other debris will “explode” under IR excitation and needs to be captured to remove from final datasets). Mount tissue in BA:BB in 3D printed sample holders and use a coverglass bottom dish as a lid for the sample. The coverglass bottom dish is filled with glycerol to match the refractive index of the sample.
