Histobond microscope slides

The third and fourth mammary gland pairs were excised from mice and fixed for downstream processing. For immunostaining, mammary glands were fixed in 4% paraformaldehyde (PFA) at room temperature for 4 hr or at 4ºC overnight, and then transferred to 70% ethanol, prior to embedding in paraffin. Alternatively, glands were processed for whole mount staining, as described below, prior to embedding in paraffin. Five micron sections were cut using a Shandon Finesse 325 Manual Microtome (Thermo Scientific) and adhered to HistoBond microscope slides (VWR).

Brain sections were cryoembedded in OCT from which they were sliced (10mm thick using a Leica Cryostat set at: −14°C for the sample and −12°C for the blade) and placed onto Histobond microscope slides (VWR 16004–416). They were immediately immersed in 100% methanol (30 sec) to help peel away the OCT. Samples were rehydrated in 75%, 50%, 25% and 0% ethanol in PBS (2 min each). Tissue were fixed in freshly prepared 4% PFA (15 min at RT in the dark), then incubated with 3 washes of 100mM Glycine in PBS pH 7.2 (3 washes, 5 min each). The brain sections were subsequently left untreated or stained with 0.1% Sudan Black B in 70% ethanol or 1X TrueBlack® for 10min. Slides were coated with Vectashield+DAPI, covered by a #1.5 coverslip, sealed with clear nail polish and stored (−20°C). Slides were imaged using a Zeiss 710 confocal microscope at 1 A.U., using either 20x air, N/A=0.8, 40x water N/A=1.2 or 100x oil N/A=1.4 lenses and imaged in the FITC (green), Cy3 (red), and Cy5 (far-red) channels. Autofluorescent perinuclear foci in either of these channels that are quenched by either Sudan Black B or True Black are indicative of lipofuscin granules.