Bx61vs light microscope

Tissue sections (5 μm, Leica RM2165) were placed in buffer at 70 °C for 3 h, diaphanized in xylol, re-hydrated and rinsed with distilled water, then heated at 90 °C immersed in a buffer citrate solution in a micro-oven. Endogenous peroxidase activity was blocked by hydrogen peroxide (H₂O₂, 3%) and blocked with normal horse serum (Vector Labs, Burlingame, CA, USA). Sections were incubated with primary antisera raised against P450arom anti-rabbit/mouse (1/400- ab18995, Abcam, Cambridge, MA, USA), NADPH-cytochrome P450 oxido-reductase (CPR) anti-mouse, rat, sheep, rabbit, guinea pig, hamster, cow, dog, human, pig, monkey (1/200- ab13513, Abcam, Cambridge, MA, USA), and P450c17 (1/200- Dr. Alan J. Conley, UC, Davis, California, USA), for 16 h. The primary antiserum was omitted for negative controls. Sections were rinsed, incubated with secondary anti-mouse/rabbit antisera ready-to-use (Immpress Universal Kit™,Vector Labs, Burlingame CA USA) followed by amplifier solution and developed with DAB (ImmPACT™ DAB, Vector Labs, Burlingame, CA USA). Sections were counter-stained with hematoxylin and mounted with coverslips. Images were captured with camera Olympus UTVO.5XC mounted on an Olympus BX61VS light microscope.

For the 3D reconstructions, we selected a single specimen of the embryonic stages X, XIII and for 0, 5, 10, 20, and 35 post-hatching days each. The samples were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer for several days. Post-fixation was made in osmium tetroxide (1% in cacodylate buffer) and after dehydration, in a graded acetone series, the samples were embedded in epoxy resin following standard protocols. Semithin section series (1.5 μm) were made with an ultramicrotome (RMC MT 7000) with a diamond knife according to the protocol established by Ruthensteiner (2008 (link)). The sections were mounted on glass slides and stained with Richardson blue (Figure 1A). Photographs of the complete microscope slides were obtained with an automated Olympus BX61VS light microscope (20x objective) equipped with an Olympus IX2-FCB digital camera and dotSlide system. Using Olympus OlyVIA software (Leadtools, USA) a snapshot of every fourth slice was taken for further digital imaging.

A FT from the temporal region (Fig. 1C) of a male Calumma crypticum (ZSM 503/2014, stored in 70% ethanol) was dissected with a razorblade. The excised FT was washed in 0.1 M phosphate buffer, stained in buffered 1% Osmium tetroxide (OsO₄) for one hour on ice, dehydrated in a graded acetone series, embedded in epoxy resin^{44 (link)}, serial sectioned in 279 planes à 1.54 µm using a RMC MT-7000 ultramicrotome with a Diatome Histo Jumbo diamond knife, mounted on glass slides, and partly stained with a 1:1 mixture of methylene blue and Azure II for approx. 5 s at 80 °C^{45 (link)}. Unstained sections (Supplementary Fig. S5A,B) were sealed under coverslips with DPX mounting medium (Agar Scientifics). The glass slides with slice-ribbons were imaged in bright field illumination using an Olympus BX61VS light microscope with UPlanSApo 10 × NA 0.4 objective and CX10 digital camera, and the program VS-ASW FL (Olympus v 2.7) for virtual slide acquisition. Images of single slices were then extracted with OlyVIA software (Olympus v 2.9; 1267 × 2119 px, 24 bit RGB, 0.98 µm/px) for subsequent volume rendering. In addition selected slices were photographed using an Olympus CX 41 light microscope with a DP25 digital camera (objectives: Olympus Plan C 10 × NA 0.25, Olympus UPlanSApo 40 × NA 0.95, and Olympus UPlanSApo 60 × NA 1.2 W); for details see Supplementary Table S8.