Acid-Free Staining Procedure to Demonstrate Nerves in Whole Vertebrate Specimens with the Differentiation of Bone and Cartilage
The value of clearing and staining whole organisms to study vertebrate anatomy is unquestionable. These methods have been developed for over a century leading to protocols to prepare triple-stained specimens to differentiate between bones, cartilage, and nerves. Despite their potential to advance the field of comparative anatomy, nerve-staining methods have been used by a small number of vertebrate systematists in part because of the inconsistently successful preparations. Here, we report on several modifications to the current Sudan black B protocols and propose a new acid-free protocol to differentiate among bone, cartilage, and nerves in whole small vertebrates. This method may also be used to stain solely bone and cartilage by eliminating the nerve-staining steps. The technique herein described is successful for preparing juveniles and adults (including miniatures).
Head regions, anterior to the left, of cleared and triple-counterstained fish specimens for bone, cartilage, and nerves using the technique herein described. (A) Stenogobius genivittatus, LIRP 16424, 53.7 mm SL, left lateral view. (B) Anchoa marinii, LIRP 4365, 1, 87.4 mm SL, left lateral view. (C) Salminus hilarii, LIRP 5911, 48.1 mm SL, left lateral view. (D) Hoplerythrinus unitaeniatus, LIRP 3945, 59.2 mm SL, left lateral view. (E) Brachyhypopomus pinnicaudatus, LIRP 6055, 120.5 mm SL, left lateral view. (F) Trichomycterus paolence, LIRP 8274, 70.6 mm SL, dorsal view. Black bars = 2 mm. Red arrows indicate nerve branches.
Specimens of Tetrapoda cleared and triple-counterstained for bone, cartilage, and nerves using the technique herein described. Hemidactylus mabouia, CHRP 2238, 67.8 mm SVL: (A) dorsal view of the head, anterior up; (B) ventral view of the head, lower jaw removed, anterior up; (C) dorsal view of the lower jaw, anterior up; (D) dorsal view of the right anterior limb, anterior down. Bokermannohyla hylax, CHRP 2236, 38.4 mm SVL: (E) dorsal view of the head, anterior up; (F) ventral view of the head, lower jaw removed, anterior up; (G) dorsal view of the lower jaw, anterior up; (H) dorsal view of the left anterior limb, anterior down. Eurycea bislineata, CHRP 2239, 34.6 mm SVL: (I) dorsal view of the head, anterior up; (J) ventral view of the head, lower jaw removed, anterior up; (K) dorsal view of the lower jaw, anterior up; (L) dorsal view of the left anterior limb, anterior down. Black bars = 2 mm. Red arrows indicate nerve branches.
Cleared and triple-counterstained juvenile (A–E) and miniature (F) specimens for bone, cartilage, and nerves using the technique herein described. (A) Bokermannohyla hylax, CHRP 2235, 34.3 mm TL, dorsal view, anterior to left. (B) Bokermannohyla hylax, CHRP 2234, 25.1 mm TL, dorsal view, anterior to left. (C) Syacium papillosum, LIRP 16677, 26.9 mm SL, ocular side view, anterior to left. (D) Prochilodus argenteus, LIRP 16924, 40.5 mm SL, left lateral view of the head, anterior to left. (E) Polypterus senegalus, LIRP 15711, 57.4 mm SL, left lateral view of the head, anterior to left. (F) Potamoglanis hasemani, LIRP 7397, 14.3 mm SL, dorsal view of the head, anterior to left. Black bars = 2 mm. Red arrows indicate nerve branches.
Contributor Notes
Associate Editor: W. L. Smith.