13-0700

antibody from Invitrogen Antibodies

Targeting: NEFM NEF3, NF-M, NFM

Provider product page for 13-0700

Immunocytochemistry

Immunohistochemistry

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Antibody data

Antibody Data
Antigen structure
References [49]
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Validations
Immunocytochemistry [2]
Immunohistochemistry [5]
Other assay [16]

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Product number: 13-0700 - Provider product page
Provider: Invitrogen Antibodies
Product name: NEFM Monoclonal Antibody (RMO-270)
Antibody type: Monoclonal
Antigen: Other
Description: The cloning partner for this antibody is Sp/2.
Antibody clone number: RMO-270
Concentration: 0.5 mg/mL

NEFM protein structure - 13-0700 shown in red.

Supportive validation

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Experimental details: Knockout of NEFM was achieved by CRISPR-Cas9 genome editing. Immunofluorescence analysis was performed on wild type HEK-293 cells (panel a,d), HEK-293 Cas9 cells (panels b,e) and HEK-293 NEFM KO cells (panel c,f). Cells were fixed, permeabilized, and labelled with NEFM Monoclonal Antibody (RMO-270) (Product # 13-0700) (5 µg/mL), followed by Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723) (1:2000). Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415) (1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal (panel c,f) upon CRISPR mediated knockout (KO) confirms that antibody is specific to NEFM (green). The images were captured at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescence analysis of NF-M was performed using 70% confluent log phase HEK-293 and HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. HEK-293 cells were labeled with NF-M Mouse Monoclonal Antibody (Product # 13-0700) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415). Panel d represents the merged image of HEK-293 showing cytoskeletal (intermediate filaments) localization. Panel e represents the merged image of HeLa cells showing no expression for NF-M protein. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of the neurofilament medium chain showing staining in the filaments of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Neurofilament medium chain monoclonal antibody (Product # 13-0700) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of the neurofilament medium chain showing staining in the filaments of paraffin-embedded rat brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Neurofilament medium chain monoclonal antibody (Product # 13-0700) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of the neurofilament medium chain in paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). Tissue sections were deparaffinized with xylene, and rehydrated with ethanol. To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were washed with water and PBS, and then blocked in 0.3% BSA for 30 min at room temperature. Tissues were then probed with a neurofilament medium chain monoclonal antibody (Product # 13-0700) in 0.3% BSA at a dilution of 1:20 for 1 hour at 37°C. Tissues were then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight 488 conjugate for 1 hour at 37°C (green). Nuclei (blue) were stained with DAPI. Images were taken at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of the neurofilament medium chain in paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). Tissue sections were deparaffinized with xylene, and rehydrated with ethanol. To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were washed with water and PBS, and then blocked in 0.3% BSA for 30 min at room temperature. Tissues were then probed with a neurofilament medium chain monoclonal antibody (Product # 13-0700) in 0.3% BSA at a dilution of 1:20 for 1 hour at 37°C. Tissues were then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight 488 conjugate for 1 hour at 37°C (green). Nuclei (blue) were stained with DAPI. Images were taken at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of the neurofilament medium chain showing staining in the filaments of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Neurofilament medium chain monoclonal antibody (Product # 13-0700) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 Cranial nerve abnormalities in Sprouty mutants. Anti-neurofilament immunohistochemistry revealing developing cranial nerves of wildtype (A), Spry1 -/- (B), Spry2 -/- (C), Spry1 +/- ;Spry2 +/- (D) and Spry1 -/- ;Spry2 -/- (E,E') E10.5 embryos is shown. All embryos were between 34 and 36 somite stage. Standard labelling of the cranial nerves was used, trigeminal ganglion (V) with opthalmic (Vo), maxillary (Vmx) and mandibulary (Vm) branches, facial nerve (VII); vestibulocochlear nerve (VIII); glossopharyngeal nerve (IX) and the vagus nerve (X). Arrows highlight abnormal morphology and asterisks indicate missing portions. The majority of developing cranial nerves present in Spry1 -/- (B), Spry2 -/- (C) and Spry1 +/- ;Spry2 +/- (D) embryos were comparable to wildtype and the latter genotype was used as a control in this study. Spry1 -/- ;Spry2 -/- embryos have trigeminal defects (e.g. absent ophthalmic branch of the trigeminal nerve in E), facial nerve defects and glossopharyngeal and vagus cranial nerves display incomplete or irregular bridging between proximal and distal ganglia (E,E'). (F-M) Neurofilament (RMO-270) and Phox2b immunohistochemistry counter stained with DAPI on sections from E10.5 Spry1 +/- ;Spry2 +/- control (F-I) and Spry1 -/- ;Spry2 -/- (J-M) mutant embryos. Neurofilament staining is indicated with green fluorescence, Phox2b labelling of motor and sensory neuron nuclei in red and DAPI stained nuclei in blue. Labelling of markers and genotypes

Supportive validation

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Experimental details: Fig. 6 Spry1-/-;Spry2-/- epibranchial cranial nerve phenotype is partially rescued by Fgf8 heterozygosity. (A-D) Anti-neurofilament immunohistochemistry showing the developing cranial nerves in Spry1 +/- ;Spry2 +/- , Spry1 -/- ;Spry2 -/- and Spry1 -/- ;Spry2 -/- ; Fgf8 +/- embryos. Note the increased glossopharyngeal nerve fibres in Spry1 -/- ;Spry2 -/- ; Fgf8 +/- embryos (red arrows in C and D) compared to the Spry1 -/- ;Spry2 -/- embryo (B). (E-G) Epibranchial placodes in E9.5 embryos as revealed by a Ngn2 antisense RNA probe. Note the enlarged geniculate (red arrow) and absence of petrosal and nodose placodes (red stars) in the Spry1 -/- ;Spry2 -/- embryo (F), and the rescue of these phenotypes in the Spry1 -/- ;Spry2 -/- ; Fgf8 +/- embryo, especially the presence of the petrosal and nodose placodes (red arrows in G).

Supportive validation

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Experimental details: Figure 7 5-HT immunoreactive axons are more abundant 9 weeks after enzyme treatment than in control mice. One ul ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal to this site in mice with severe compression injury. After 9 weeks, the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence. Double immunostaining for serotonin (5-HT) and neurofilament-M (NF-M) shows higher immunoreactivities caudal injury site in the ChaseABC ( D,E,F ) and ARSB ( G,H,I ) treated mice versus buffer treated control mice ( A,B,C ). ( A,D,G ) Immunostainings for 5-HT and ( B,E,H ) NF-M, and ( C,F,I ) merged for 5-HT with NF-M. 5-HT immunoreactive axons are seen beyond the injury site in the ChaseABC and ARSB injected mice. Immunoreactive areas were quantified above threshold using Image J software ( J ). Mean fluorescence intensity of the area between the injury site and 1 mm caudal to it is significantly higher in ChaseABC treated versus buffer treated control mice. Asterisk indicates significant differences between the groups *p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8 Tyrosine hydroxylase (TH) immunoreactive axons are more abundant 9 weeks after enzyme treatment than in control mice. One ul ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal of this site in mice with severe compression injury. After 9 weeks, the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence. Double immunostaining for TH and neurofilament-M (NF-M) shows higher immunoreactivities caudal to the injury site in the ChaseABC ( D,E,F ) and ARSB ( G,H,I ) treated mice versus the buffer treated control mice ( A,B,C ). ( A,D,G ) Immunostainings for TH and ( B,E,H ) NF-M, and ( C,F,I ) merged for TH with NF-M. Immunoreactive areas were quantified above threshold using Image J software ( J ). Mean fluorescence intensity of the area between the injury site and 1 mm caudal to it shows significantly higher immunofluorescence intensity in ARSB versus buffer treated control and ChaseABC treated mice. Asterisks indicate significant differences between the groups *p

Supportive validation

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Experimental details: Fig. 1. Neural crest cells form corridors associated with sensory neuroblasts in chick and mouse. ( A , B ) Lateral view of HH15 + chick embryo showing association of NCCs with neuroblasts in (A) a 3D reconstruction of HNK1 (magenta) and NFM (green) staining, and (B) a schematic representation (NCCs, orange; neuroblasts, blue; general NCC territory is shown by an orange line; the direction of migration is indicated by arrows). ( C , D ) HH17 transverse section (C) and digital coronal re-section (D) reveal HNK1 + NCCs (magenta) peripheral to ISL + neuroblasts (green) (single optical confocal section). ( E ) NCC labelling by GFP electroporation confirms NCCs (green) encircle NFM + neuroblasts (magenta); HH18 coronal section. ( F ) Following placode electroporation, bipolar GFP + cells (green) are seen migrating within the HNK1 + NCC corridor (magenta) (sagittal, HH17, single optical confocal section). ( G ) Lateral view of 29 ss Wnt1cre:R26RYFP mouse embryo shows the association of NCCs (green) with NFM + neuroblasts (red). ( H , H ') Transverse section (H) and digital coronal re-section (H') show YFP + NCCs (magenta) encircling NFM + neuroblasts (green) as in chick (confocal stack projection). Asterisks indicate placodes; I, II and III, pharyngeal arches 1, 2 and 3; NT, neural tube; OV, otic vesicle; p, petrosal; g, geniculate; t, trigeminal ganglia; r, rhombomere. Dotted lines in A indicate the levels of sections in C,E; dotted lines in C indicate the level of the s

Supportive validation

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Experimental details: Figure 2 Caffeine exposure impaired neurofilament (NF) and HNK-1 expression in early chick eyes. HH10 chick embryos were treated with 15 mumol/egg caffeine for 1.5 days and then whole-mount double immunostained with NF and HNK-1 antibodies. (A-D) Control and caffeine-treated embryos stained with HNK-1 (A and C) and NF (B and D) antibodies. Higher magnified bright-field images indicated the eye malformation induced by caffeine (black arrows). (E-H) Transverse sections of A and B (dotted white lines) showing the expression pattern of HNK-1 and NF in the control. There are some HNK-1 + neural crest cells surrounding the eyes in control embryo (white arrow in F), but absent in caffeine-treated ones. (I-L) Transverse sections of C and D (dotted white lines) showing HNK-1 and NF expression in caffeine-treated eyes is distinctly different from control eyes. BF, bright-field. Scale bars = 1 mm in A-D; 100 mum in E-L.

Supportive validation

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Experimental details: Fig. 1 Isolation of embryonic chick placode-derived cranial sensory neurons by dissection and FACS. A Schematic of cranial sensory ganglia in embryonic day 12 (HH38) chick (adapted from [ 6 ]). The ganglia are labelled: trigeminal ganglion as two separate lobes (Top: ophthalmic; Tmm: maxillomandibular); vestibulo-acoustic ganglion (VA); epibranchial series as three separate ganglia (G: geniculate; P: petrosal; N: nodose). Also labelled are the neural crest-derived superior- jugular ganglionic complex (S/J); the inner ear (IE); and forebrain (FB); midbrain (MB) and hindbrain (HB) of the CNS. Colours indicate the sensory modality of ganglion: blue: general somatosensory; magenta: special somatosensory; green: viscerosensory. B Representative dissections of cranial sensory ganglia: Top, Tmm and VA at HH18, and P and N at HH23. C , D Representative FACS plots of cells stained for live/dead stain and NFM. C Control cell population: limb bud cells devoid of neurons, containing 50 % dead cells. D Petrosal ganglion cell population: 36 % of cells are NFM positive and dead cell marker negative

Supportive validation

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Experimental details: Figure 1 Tissue clearing following antibody staining allows for visualization of axons throughout entire rat sciatic nerves. Tissue were stained with anti-neurofilament antibodies using a whole mount immunofluorescence method. (a) A lateral (xy) confocal image at the depth of approximately 400 mum from the surface of a stained and cleared wild type rat sciatic nerve. (b) Three dimensional view of a whole stained and cleared sciatic nerve. Non-specific GFP epineurial staining can also be seen secondary to presence of fibroblasts, collagen, and vasa nervorum in the epineurium, which does not affect analysis. (c) Reconstructed transverse cross sectional image. Three fascicles surrounded by the epineural sheath can be recognized. For comparison, a transverse cross sectional image of non-tissue cleared, stained sciatic nerve is shown in (d); note that non-specific staining occurs in the epineurial sheath as well, but only the right portion of the sheath is observed. (e) Two-photon transverse cross sectional image of a sciatic nerve from a transgenic rat (thy-1 GFP).

Supportive validation

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Experimental details: Figure 2 Deep tissue and high resolution images of tissue cleared nerves by confocal microscopy following anti-neurofilament antibody labeling. (a) Lateral (xy) confocal image. (b) Cross sectional image. The elongated shape of the axons in the z direction is caused by the lower relative axial resolution of confocal microscopy.

Supportive validation

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Experimental details: Impact of preventive BLZ945 treatment on microglia and neurodegeneration in chronic CPZ-induced demyelination. (a) Experimental procedure. (b) Immunohistochemical studies of Iba-1 in the cortex (CX), corpus callosum (CC), external capsule (EC), and striatum (ST). Quantification of Iba-1+ cells. (c) High magnification confocal images of Iba-1+ cells in CX and CC to evaluate morphological changes. (d) Immunohistochemical studies of Iba-1 and MBP in CC. Quantification of Iba-1+ cells, MBP+Iba-1+ cells and MBP+Iba-1+/Iba-1+ ratio in CC. (e) Immunohistochemical studies of MBP and NFM in the CX. (f) Quantitative colocalization assessment by Mander's coefficient . (g) Quantification of Neurotrace+ cells, AbetaPP+ cells and AbetaPP+ /Neurotrace+ ratio. (h) Immunohistochemical studies of AbetaPP+ cells in cingulum (CL) and CC. Values represent the mean +- SD of three different mice: * p < .05, *** p < .001 as compared with C; ### p < .001 as compared with BLZ945; ++ p < .01, +++ p < .001 as compared with CPZ using one-way ANOVA followed by Bonferroni post hoc tests [Color figure can be viewed at wileyonlinelibrary.com ]

Supportive validation

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Experimental details: Effect of therapeutic BLZ945 treatment on chronic CPZ-induced demyelination. (a) Experimental procedure . (b) Immunohistochemical studies of MBP in the cortex (CX) . Quantification of MBP immunoreactivity and IOD in the CX, corpus callosum (CC), external capsule (EC), and striatum (ST) . (c) Immunohistochemical studies of GFAP in the CX. Quantification of GFAP+ cells in the CX, CC, EC and ST . (d) Immunohistochemical studies of Iba-1 in the CX. Quantification of Iba-1+ cells in the CX, CC, EC and ST. (e) Immunohistochemical studies of MBP and NFM in the CX. Quantitative colocalization assessment by Mander's coefficient. (f) Immunohistochemical studies of AbetaPP+ cells. Quantification of Neurotrace+ cells, AbetaPP+ cells and AbetaPP+/Neurotrace+ ratio. Values represent the mean +- SD of three different mice: * p < .05, ** p < .01, *** p < .001 as compared with C; # p < .05, ## p < .01, ### p < .001 as compared with BLZ945; + p < .05, +++ p < .001 as compared with CPZ using one-way ANOVA followed by Bonferroni post hoc tests [Color figure can be viewed at wileyonlinelibrary.com ]

Supportive validation

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Experimental details: Figure 4 Late subtype specification is altered and boundary cap cell identity is lost in mutant animals. (A) Illustration of a transverse section at E11.5 displaying cells, which contribute to the DRG in red. Green box represents caption area for subfigures B-G. (B-G) Triple immunohistochemistry at E11.5 for beta-gal expressed by the R26R reporter and for markers for subtype specification of sensory neurons: tyrosine receptor kinases RET and TrkA, TrkB and TrkC. Dashed lines surround the border of R26R -positive cells contributing to the DRG. (H) Quantification of late sensory subtypes per DRG displays an altered distribution of sensory neuron subtypes in mutant animals. (I) Illustration of a transverse section at E12.5 displaying boundary cap cells in red. Green box represents caption area for subfigures J-O, respectively. (J-L) Double immunohistochemistry for Krox20 as a marker for differentiated boundary cap cells and neurofilament (NF) at E12.5. (M-O) Triple immunohistochemistry for Krox20, NF and Islet1/2 as a marker for motor neurons at E12.5. (J',M') In control animals, sensory axons invading the dorsal entry zone of the neural tube and motor axons penetrating the prospective motor exit point of the neural tube are surrounded by boundary cap cells. (K',L',N',O') Dorsal as well as ventral Krox20 expression is lost in both mutant animals. Arrows show migrating motor neurons, which are exiting or have already exited the ventral neural tube du

Supportive validation

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Experimental details: Figure 7 Development of boundary cap cells depends on neural tube-derived Wnt. (A, C) Illustration of a transverse section at E10.5 (A) and E12.5 (C) displaying cells contributing to the DRG in red. Green box represents caption area for subfigures B and D. respectively. (B, D) Double immunohistochemistry for Wls and beta-gal expressed in Wnt1-Cre R26R mice shows that mesenchymal tissue and not cells populating the DRG express Wls. (B', D') Corresponding single fluorescence channels of the same sections. Dashed lines frame lineage-traced cells. (E, I) Illustration of a transverse section at E12.5 displaying BCCs in red. Green box represents caption area for subfigures F-H and J-M, respectively. (F-H) Double immunohistochemistry for Krox20 and neurofilament (NF). (F'-H') Corresponding single fluorescence channels of the same sections. (G') Dorsal BCCs are lost upon conditional knock-out of Wls using Wnt1-Cre . (H') However, BCCs are rescued in conditional knockout of Wls using Sox10-Cre . (J-M) Double immunohistochemistry for Wls and beta-gal expressed by the Cre reporter line R26R . J'-M' and J""-M"" are higher magnifications of the boxed areas in J-M, respectively. Dashed lines frame sensory axons . (J-J"", L-L"") Wls is expressed at the site where sensory axons enter the dorsal neural tube at the dorsal entry zone and strongly expressed in the roofplate (arrow, J, L) of control animals. (K-K"") Expression of Wls is lost in dorsal neura

Supportive validation

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Experimental details: Figure 6 AAPH exposure replicates the effect of caffeine on the developing eye. Early chick embryos exposed to 10 mumol/egg AAPH for 6 days and immunostained with NF antibody. (A and B) Control (A) and AAPH-treated (B) chick embryos. (C-E) NF immunostaining demonstrates the presence of neurons in control (C) and AAPH-treated (right eye, D) and (left eye, E) lens. The staining shows AAPH-treatment repressed neuron development in the eyes. (C'-E''') Transverse section of the lens at the levels indicated by white line in A and B. Bright-field (C', D', E'), NF immunostaining (C'', D'', E'') and NF immunostaining + DAPI (C''', D''', E'''). (O and P) Bar charts showing AAPH significantly increased the incidence of eye malformation (O) and reduced the size (diameter) of the eyes (P). NF, neurofilament. Scale bars = 2 mm in A and B; 1 mm in C-E; 200 mum in F-N.