, the developmental stage of a zebrafish can be measured by its standard length ( sl ) , or the straight line distance in millimeters from the anteriormost point of the upper lip to the base of the caudal fin ( posteriormost hypural plate ) . at approximately one month post fertilization ( 10 - 12 mm sl ) , the nasal and maxillary barbels appear as transparent epithelial buds near the olfactory pits and on the posterior corners of the maxillae , respectively . as the zebrafish matures , the barbels extend into narrow , whisker - like appendages . because the maxillary barbel is larger ( 2 - 3 mm ) and easier to manipulate , our protocol applies only to this particular appendage ; similar techniques , however , could be adapted to the smaller nasal barbel . anaesthetize adult zebrafish at the desired stage ( e.g. , 1.5 - 2.5 cm sl , ~3 - 6 months post - fertilization ) by immersion in 0.015% ms-222 ( ethyl 3-aminobenzoate methanesulfonate ) buffered to ph 7.0 in system water . depending on fish size , complete anesthesia takes approximately 2 - 5 minutes . using an aquarium fishnet , transfer 1 - 3 fish at a time to a folded piece of wet paper towel in a petri dish . using a blunt spatula , orient the fish so they are parallel to each other and left lateral side up . fold part of the wet paper towel over the caudal part of the fish , covering the entire body up to the operculum ( gill covers ) to keep the skin and gills moist during the procedure . view the petri plate under the stereomicroscope , using low - angle incident light to illuminate the head region . focus on the base of the left maxillary barbel , which protrudes from the posterior ventral corner of the maxilla ( fig . tightly grasp the shaft of the barbel slightly distal to the edge of the maxilla . elevate the barbel shaft and insert the jaws of a fine - tipped spring scissors just proximal to the forceps . slide the jaws of the scissors along the shaft of the barbel until the cutting edge approaches the edge of the maxilla . the amputated barbel , still gripped in the forceps , can be removed for fixation or further observation . immediately transfer the fish to a small tank of clean system water ( ~500 ml ) containing one drop of methylene blue to control superficial fungal infection . allow the fish to recover overnight . the next morning , return the fish to the rearing system . collect fish by appropriate euthanasia technique and fix in a 50 ml tube of 4% paraformaldehyde - phosphate buffered saline ( pbs ) with agitation at 4c overnight . after fixation , dispose of paraformaldehyde waste in an approved container and rinse the tissue thoroughly in several changes of pbs . prepare in a 250 ml glass flask 150 ml of 2% agarose ( tm ~37c ) in distilled water . equilibrate the molten solution in a 50 - 60c water bath . assemble a standard gel electrophoresis rig by placing a small gasketed gel mold ( 10 x 10 cm ; ~100 ml gel volume ) firmly against the sides of the buffer chamber . add 2 - 4 small - toothed sample combs ( 4 x 1.5 mm ) .   on a level surface , pour the melted agarose into the mold to just cover the base of the combs , forming very shallow wells . immediately put the leftover agarose back into the warm water bath ; this will be used later to embed the tissue ( steps 9 - 12 ) . put a long glass pasteur pipette into the flask to keep the pipette to the same temperature as the agarose . remove the combs , then remove the gel mold containing the hardened gel from the buffer chamber . firmly tape the open sides of the gel mold with laboratory tape so the gel does not slide out . this tape should extend several millimeters over the surface of the agarose so that additional agarose can be added later ( step 13 ) . magnify an empty well and bring the edges into focus .   onto the surface of the agarose adjacent to the well , pipette a drop of water or buffer containing the fixed barbel specimens ( e.g. , regenerate and control ) . using bent insect pins , orient the barbel shafts parallel to each other and aligned against opposite long edges of the well . when ready to embed , use a fine pipette tip or the corner of a laboratory tissue to remove excess fluid from the well . using the warmed glass pasteur pipette , gently pipette fresh molten agarose in and around the specimens to seal the barbel tissue in place . do not overfill . before the agarose hardens , place a numbered paper specimen label next to the well and secure it with a small drop of agarose . if desired for image calibration , insert a piece of paper with a printed micrometer scale ( e.g. , 
 http://incompetech.com/graphpaper/multiwidth/ ) into the bottom of an empty well . flatten the paper to the bottom of the well and cover it with warm agarose . after all of the specimens are embedded , the surface of the gel will be irregular , with hardened drops of agarose . place the gel on a flat surface and gently pour in additional melted agarose until the upper surface is uniform . use the smallest amount of agarose necessary to obtain a smooth surface ; too much agarose will obscure transmitted - light photography . allow this top layer to harden . the gel can now be photographed on the stage of a stereomicroscope to record gross morphology for each barbel pair . the entire gel can be stored wrapped in wet paper towels and sealed in a plastic bag at 4 for several weeks .   for further analysis , agar blocks containing matched pair of barbels can be cut out of the gel with a scalpel or razor blade and processed for paraffin histology or cryosectioning by standard methods . alternatively , individual barbels can be dissected out of the agarose with fine needles , rinsed in water , and processed for other downstream applications ( e.g. , whole - mount immunohistochemistry or confocal microscopy ) . , the developmental stage of a zebrafish can be measured by its standard length ( sl ) , or the straight line distance in millimeters from the anteriormost point of the upper lip to the base of the caudal fin ( posteriormost hypural plate ) . at approximately one month post fertilization ( 10 - 12 mm sl ) , the nasal and maxillary barbels appear as transparent epithelial buds near the olfactory pits and on the posterior corners of the maxillae , respectively . as the zebrafish matures , the barbels extend into narrow , whisker - like appendages . because the maxillary barbel is larger ( 2 - 3 mm ) and easier to manipulate , our protocol applies only to this particular appendage ; similar techniques , however , could be adapted to the smaller nasal barbel . anaesthetize adult zebrafish at the desired stage ( e.g. , 1.5 - 2.5 cm sl , ~3 - 6 months post - fertilization ) by immersion in 0.015% ms-222 ( ethyl 3-aminobenzoate methanesulfonate ) buffered to ph 7.0 in system water . depending on fish size , complete anesthesia takes approximately 2 - 5 minutes . using an aquarium fishnet , transfer 1 - 3 fish at a time to a folded piece of wet paper towel in a petri dish . using a blunt spatula , orient the fish so they are parallel to each other and left lateral side up . fold part of the wet paper towel over the caudal part of the fish , covering the entire body up to the operculum ( gill covers ) to keep the skin and gills moist during the procedure . view the petri plate under the stereomicroscope , using low - angle incident light to illuminate the head region . focus on the base of the left maxillary barbel , which protrudes from the posterior ventral corner of the maxilla ( fig . tightly grasp the shaft of the barbel slightly distal to the edge of the maxilla . elevate the barbel shaft and insert the jaws of a fine - tipped spring scissors just proximal to the forceps . slide the jaws of the scissors along the shaft of the barbel until the cutting edge approaches the edge of the maxilla . the amputated barbel , still gripped in the forceps , can be removed for fixation or further observation . immediately transfer the fish to a small tank of clean system water ( ~500 ml ) containing one drop of methylene blue to control superficial fungal infection . collect fish by appropriate euthanasia technique and fix in a 50 ml tube of 4% paraformaldehyde - phosphate buffered saline ( pbs ) with agitation at 4c overnight . after fixation , dispose of paraformaldehyde waste in an approved container and rinse the tissue thoroughly in several changes of pbs . prepare in a 250 ml glass flask 150 ml of 2% agarose ( tm ~37c ) in distilled water . equilibrate the molten solution in a 50 - 60c water bath . assemble a standard gel electrophoresis rig by placing a small gasketed gel mold ( 10 x 10 cm ; ~100 ml gel volume ) firmly against the sides of the buffer chamber . add 2 - 4 small - toothed sample combs ( 4 x 1.5 mm ) .   on a level surface , pour the melted agarose into the mold to just cover the base of the combs , forming very shallow wells . immediately put the leftover agarose back into the warm water bath ; this will be used later to embed the tissue ( steps 9 - 12 ) . put a long glass pasteur pipette into the flask to keep the pipette to the same temperature as the agarose . remove the combs , then remove the gel mold containing the hardened gel from the buffer chamber . firmly tape the open sides of the gel mold with laboratory tape so the gel does not slide out . this tape should extend several millimeters over the surface of the agarose so that additional agarose can be added later ( step 13 ) . magnify an empty well and bring the edges into focus .   onto the surface of the agarose adjacent to the well , pipette a drop of water or buffer containing the fixed barbel specimens ( e.g. , regenerate and control ) . using bent insect pins , drag the moist specimens into the well and push them to the bottom . orient the barbel shafts parallel to each other and aligned against opposite long edges of the well . when ready to embed , use a fine pipette tip or the corner of a laboratory tissue to remove excess fluid from the well . using the warmed glass pasteur pipette , gently pipette fresh molten agarose in and around the specimens to seal the barbel tissue in place . do not overfill . before the agarose hardens , place a numbered paper specimen label next to the well and secure it with a small drop of agarose . if desired for image calibration , insert a piece of paper with a printed micrometer scale ( e.g. , 
 http://incompetech.com/graphpaper/multiwidth/ ) into the bottom of an empty well . flatten the paper to the bottom of the well and after all of the specimens are embedded , the surface of the gel will be irregular , with hardened drops of agarose . place the gel on a flat surface and gently pour in additional melted agarose until the upper surface is uniform . use the smallest amount of agarose necessary to obtain a smooth surface ; too much agarose will obscure transmitted - light photography . allow this top layer to harden . the gel can now be photographed on the stage of a stereomicroscope to record gross morphology for each barbel pair . the entire gel can be stored wrapped in wet paper towels and sealed in a plastic bag at 4 for several weeks .   for further analysis , agar blocks containing matched pair of barbels can be cut out of the gel with a scalpel or razor blade and processed for paraffin histology or cryosectioning by standard methods . alternatively , individual barbels can be dissected out of the agarose with fine needles , rinsed in water , and processed for other downstream applications ( e.g. , whole - mount immunohistochemistry or confocal microscopy ) . the zebrafish maxillary barbel is an underutilized tissue system for studying the growth , maintenance , and regeneration of several cell types in zebrafish . although the barbel appendage has no human analog , the cell types it contains are highly conserved , making it possible to study skin , glands , melanocytes , circulatory vessels and nerves in an optically clear and anatomically simple cylindrical structure . similar to the well - studied caudal fin , barbel tissue can be induced to regenerate by amputation . using the border of the maxilla as an anatomical landmark , the amputation plane can be placed precisely , facilitating the measurement of barbel regrowth . for an experienced operator recovery is rapid , and we have so far detected no short or long - term effects on fish behavior . zebrafish with one maxillary barbel swim , eat and breed as effectively as non - surgical controls , and have comparable survival to the time of tissue collection , up to 6 months after surgery . the physiological impact of this surgery predicted to be minimal because 1 ) the extraoral taste buds carried on the barbel are also found on many other parts of the fish epithelium , including the lips , cheeks and head , and 2 ) differentiating taste buds appear on the regenerating barbel within 72 hours ( leclair et al . , unpublished data . ) this makes the maxillary barbel a minimally invasive system for studying wound healing , revascularization , and reinnervation within the context of an adult vertebrate . after surgical induction of regeneration , barbels can be collected at intervals for morphometric measurement and/or microscopic analysis of fixed tissue . conveniently , the maxillary barbel is approximately the length ( 2 - 3 mm ) and diameter ( 100 - 200 mm ) of a zebrafish embryo , facilitating the application of many standard protocols , including paraffin histology , cryosectioning , whole - mount immunohistochemistry , and in situ hybridization . taken together , these features make the maxillary barbel a highly feasible in vivo model for studying tissue repair and regeneration .
barbels are skin sensory appendages found in fishes , reptiles and amphibians . 
 the zebrafish , danio rerio , develops two pairs of barbels- a short nasal pair and a longer maxillary pair . 
 barbel tissue contains cells of ectodermal , mesodermal and neural crest origin , including skin cells , glands , taste buds , melanocytes , circulatory vessels and sensory nerves . unlike most adult tissue , 
 the maxillary barbel is optically clear , allowing us to visualize the development and maintenance of these tissue types throughout the life cycle . 
 this video shows early development of the maxillary barbel ( beginning approximately one month post - fertilization ) and demonstrates a surgical protocol to induce regeneration in the adult appendage ( > 3 months post - fertilization ) . 
 briefly , the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla . a fine 
 , sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level , establishing an anatomical landmark for the amputation plane . 
 regenerative growth can be measured with respect to this plane , and in comparison to the contralateral barbel . 
 barbel tissue regenerates rapidly , reaching maximal regrowth within 2 weeks of injury.techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels ( regenerate and control ) in the wells of a standard dna electrophoresis gel 
 . embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry , and can be stored for weeks prior to downstream applications such as paraffin histology , cryosectioning , and/or whole mount immunohistochemistry . 
 these methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish .