36-1700

antibody from Invitrogen Antibodies

Targeting: F11R CD321, JAM-1, JAM-A, JAM1, JAMA, JCAM, PAM-1

Provider product page for 36-1700

Western blot

ELISA

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [77]
Comments [0]
Validations
Western blot [1]
Immunohistochemistry [1]
Other assay [39]

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Product number: 36-1700 - Provider product page
Provider: Invitrogen Antibodies
Product name: JAM-A (CD321) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 0.25 mg/mL
Storage: -20°C

F11R protein structure - 36-1700 shown in red.

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Experimental details: Figure 1 Immunocytochemical JAM-A tight junction staining in primary HBMEC is reduced under barrier-disturbing pro-inflammatory conditions. Cells were either left unstimulated or treated with TNF-alpha 10 ng/mL and IFN-gamma 100 U/mL for 24 h. A Immunocytochemical staining was performed with primary antibodies against JAM-A (M.Ab.F11), occludin and ZO-1. F-Actin was stained with Alexa Fluor 488 phalloidin. Representative for 5 independent experiments with different HBMEC preparations. B Analysis of paracellular barrier function by dextran 3000 transwell permeability assays. Mean and SD of 4 independent experiments in octuplicates with different HBMEC preparations. The two-tailed Mann-Whitney U test was used for statistical analysis. Bar = 25 um.

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Experimental details: Figure 2 In contrast to ICAM-1, JAM-A overall surface expression levels on HBMEC do not change upon pro-inflammatory stimulation. Flow cytometric analysis of Accutase(tm)-dissociated single cell suspensions of primary HBMEC after stimulation as in Figure 1 . JAM-A was stained with M.Ab.F11 and a rabbit polyclonal antibody from Zymed. Staining against ICAM-1 served as a positive control. In parallel, unstimulated and stimulated HBMEC were stained with matched isotype control antibodies. Filled histograms represent isotype stainings, open histograms JAM-A and ICAM-1 stainings. Representative for 5 independent experiments with different HBMEC preparations.

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Experimental details: Figure 3 Pro-inflammatory stimulation of HBMEC induces JAM-A dissociation from the actin cytoskeleton. A Primary HBMEC were either left unstimulated or treated with TNF-alpha 10 ng/mL or IFN-gamma 100 IU/mL alone or in combination. Cell protein extracts were generated with a Nonidet-P40 based cell lysis buffer and subjected to Western blot analysis. JAM-A was stained with M.Ab.F11. Staining of the same, peroxidase-inactivated membranes with a rabbit antibody against beta-actin served as a loading control. B N-deglycosylation of JAM-A with increasing concentrations of PNGase F for 2 h. A rabbit polyclonal antibody against JAM-A (Zymed) was used for the detection of JAM-A. The asterisk represents N-glycosylated, the open circle N-declycosylated JAM-A. Representative experiments out of at least 5 independent experiments with different EC preparations for each subpanel of the figure are shown.

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Experimental details: Figure 4 HBMEC constitutively release sJAM-A, which is not altered under inflammatory or hypoxia/reoxygenation conditions. A Representative Western blot of a HBMEC whole cell protein extract, employing the goat anti-JAM-A antibody which was used as capture antibody for a self-developed sandwich ELISA (compare Figure 3 ). B Typical standard curve of the self-developed ELISA for JAM-A detection in culture supernatants and serum. C and D Concentrations of sJAM-A in culture supernatants of primary HBMEC ( C ) or hMCEC/D3 ( D ) which were either left unstimulated for 48 h, stimulated with TNF-alpha 10 ng/mL and IFN-gamma 100 U/mL for 48 h or subjected to 1% O 2 for 24 h followed by a reoxygenation period of 24 h. Please note: cell culture supernatants were concentrated by filter centrifugation before ELISA measurement, resulting in ~10-fold increased JAM-A concentration. Mean and SD of 3 independent experiments. The Kruskal-Wallis test was used for statistical analysis. n.s., not significant. E Comparative Western blot analysis of a whole cell protein extract and a TCA-precipitated protein extract from cell culture supernatant, using a rb polyclonal antibody (Zymed) for JAM-A detection. Representative of 3 independent experiments with different HBMEC preparations. The asterisk represents full-length JAM-A, open circles represent soluble JAM-A. F Comparative Western blot analysis of JAM-A in TCA-precipitated cell culture supernatants, using the same primary antibody as in E, after

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Experimental details: Figure 7 Expression of JAM-family members in liver sinusoidal endothelial cells. (A) Immunofluorescent co-staining of rat liver cryosections with anti-JAM-A (green), anti-VE-cadherin (red), and anti-Stabilin-2 (blue) antibodies. (B) Immunofluorescent co-staining of human liver cryosections with anti-JAM-A (green), anti-CD32b (red), and anti-Stabilin-2 (blue) antibodies. Images were acquired using laser scanning confocal microscopy. Bars 11.9 um. (C) Quantitative reverse transcriptase-PCR with mRNA isolated from rat LSECs and rat LMECs (n indicates the number of samples analyzed, error bars represent SEM). Primers specific for JAM-A, JAM-B, JAM-C, and beta-Actin as normalizer were used.

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Experimental details: Figure 4. JAM-A localization is disorganized in carcinoma in situ and becomes highly expressed in malignant seminoma. Confocal immunofluorescence of normal human testis ( A ), carcinoma in situ ( B and C ) and seminoma sections ( D ) detecting JAM-A (red, asterisks) and connexin 43 (green, gap junctions, squares). Inserts are negative controls. (Bar = 50 um).

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Experimental details: Figure 2 Peritonitis is associated with gut barrier derangement (a) H&E-stained sections of ileum from mice 16 h after injection with NS, injection with 40 mg/kg LPS, sham operation, or CLP. Note abundant epithelial sloughing in animals injected with LPS or subjected to CLP. Data are representative of at least 9 animals in each group. (b) Transepithelial passage of FITC-dextran and bacteria during peritonitis. Mice were gavaged with FITC-dextran and E. coli 35354T prior to indicated treatments. Serum concentrations of FITC-dextran (top) and counts of viable E. coli in combined mesenteric lymph nodes and spleens (bottom) were determined 16 h post treatment. *, Significant differences (p = 12 in each group). (c) Localization of JAM-A (red) in ileal epithelium 16 h after indicated treatments. Note increased re-distribution of JAM-A from borders to intracellular space of enterocytes in LPS and CLP samples. (d) Localization of ZO-1 (red) in ileal epithelium 16 h after indicated treatments. Note increased redistribution of ZO-1 from TJ to intracellular space of enterocytes in LPS and CLP samples. DAPI-stained nuclei appear in blue. Bar=50 mum. All images are representative of at least 3 animals in each group.

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Experimental details: Figure 5 Effects of Celecoxib on localization of TJ-associated proteins JAM-A and ZO-1 (a) JAM-A (red) after injection with NS or LPS, with or without indicated doses (mg/kg) of Celecoxib. (b) JAM-A (red) after sham operation or CLP, with or without indicated doses of Celecoxib. (c) ZO-1 (red) after injection with NS or LPS, with or without indicated doses of Celecoxib. (d) ZO-1 (red) after sham operation or CLP, with or without indicated doses of Celecoxib. All images show sections of ileal samples taken 16 h after peritonitis-inducing treatments and are representative of at least 3 animals in each group. DAPI-stained nuclei appear in blue. Bar=100 mum.

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Experimental details: Figure 3 BTB integrity was disrupted by Shp2 deletion. ( A ) BTB permeability was assayed with the biotin tracer (red) using confocal microscopy in two-week-old mice. Asterisk indicates the biotin tracer in the adluminal compartment of the seminiferous tubules. ( B ) Immunohistochemical staining of the BTB junction proteins Cx43, Claudin11 and JAMA in seminiferous tubules in two-week-old mice. ( C ) Altered BTB-related genes in the microarray gene database, and the protein levels of various genes were analyzed by Western blotting in testes from two-week-old mice. The full-length blots are presented in Supplemental Figure S7 . ( D ) The quantity of the cell junctions between primary Sertoli cells isolated from two-week-old mice was measured by transepithelial resistance (TER) assays. The experiments were repeated at least three times, and one representative result was shown. ( E ) Shp2 expression and Erk and Akt phosphorylation in primary Sertoli cells used in the TER assays. Tubulin was used as a loading control. The full-length blots are presented in Supplemental Figure S8 . Ad, adenovirus virus; f/f, Shp2 f/f cells; ko, SCSKO cells; Q79P, constitutively active Shp2 mutant. The values are expressed as the mean +- SEM. Statistical analysis was performed using Student's t -test. Asterisks denote the statistical significance, **P < 0.01; ***P < 0.001.

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Experimental details: Figure 5 Knockdown of Rai14 in the Sertoli cell epithelium with an established TJ-permeability barrier in vitro by RNAi disrupts actin filament organization and the TJ barrier. (A) Sertoli cells cultured alone on Matrigel-coated 12-well dishes for 2-day with an established TJ-permeability barrier were transfected with Rai14 siRNA duplexes (Rai14 RNAi) versus non-targeting control duplexes (Ctrl RNAi) at 100 nM using Ribojuice transfection medium for 24 hr, thereafter, cells were washed twice and cultured in F12/DMEM for 12 hr to allow recovery. Thereafter, cells were transfected again under the same conditions for another 24 hr. Thereafter, cells were rinsed with fresh F12/DMEM and cultured for an additional 12 hr before termination, and used to prepare lysates for immunoblotting using antibodies against several BTB-associated constituent or regulatory proteins. A knockdown of Rai14 by ~50% was noted in which the control was arbitrarily set at 1 against which statistical comparison was performed (B) without any apparent off-target effects (A). The findings shown herein are the results of 3 independent experiments excluding pilot experiments which were used to establish optimal experimental conditions, such as different concentrations of siRNA duplexes and Ribojuice. It was noted that we achieved only ~50-60% knockdown of Rai14 in several pilot experiments, unlike other target genes [ e.g ., Scribble, beta1-integrin, and P-glycoprotein] wherein we could silence the target gene

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Experimental details: Figure 1 Rai14 is an actin-binding protein in the rat testis. ( A ) A study by RT-PCR to confirm the expression of Rai14 in adult rat testis, Sertoli cells (SC, isolated from 20-day-old rat testes and cultured for 4-day), and germ cells (GC, isolated from adult rat testes and cultured for 16 hr). ( B ) Immunoblotting also confirmed the expression of Rai14 in the rat testis, Sertoli and germ cells, and the relative expression of Rai14 in SC vs. GC was shown in the histogram with n = 3 experiments in which the relative expression level of Rai14 in the testis was arbitrarily set at 1 so that the relative expression level between these samples can be compared. ( C ) The specificity of the anti-Rai14 antibody ( Table 1 ) was assessed by immunoblotting using lysates of GC (20 ug protein). ( D ) Using the specific anti-Rai14 antibody, Rai14 was shown to be an actin-binding protein by co-immunoprecipitation (Co-IP); however, Rai14 did not structurally interact with any of the BTB-associated proteins including several actin-binding and regulatory proteins ( e.g ., Arp3, drebrin E, Eps8) and vimentin (an intermediate filament-based constituent protein). However, Rai14 was found to structurally interact with an actin cross-linking protein palladin which is known to be involved in conferring actin filament bundles in other mammalian cells [48] . ( E ) Rai14 (red) was also shown to be an actin-binding protein by dual-labeled immunofluorescence analysis in which it co-localized with F-acti

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Experimental details: 1 Figure Intravitreal injection of melatonin improves retinal function and maintains iBRB integrity in diabetic rat retinas. (a) Representative ERG waveforms recorded from normal control and 8-week diabetic rats treated with or without melatonin. Statistical analysis of both a-wave (b) and b-wave (c) amplitudes among the three groups ( n = 15 eyes per group). (d) Fluorescent microscopic images of retinal flat-mounts in 8-week diabetic rats and normal control injected with 10 kDa FITC-dextran. Arrows indicate retinal vascular leakage. Scale bars, 100 mum. (e) Quantification of retinal vascular permeability by FITC-dextran ( n = 3 per group). The change of claudin-5 (f), ZO-1 (g), occludin (h), and JAM-A (i) expression was examined with Western blot in 8-week diabetic rat retinas with or without melatonin. Data were expressed as mean +- SEM ( n = 9 per group); p values by one-way ANOVA. * p < .05; ** p < .01; *** p < .001 compared with diabetes group. D, diabetes; ERG, electroretinogram; iBRB, blood-retinal barrier; M, diabetic rats treated with melatonin; N, normal control

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Experimental details: 5 Figure Melatonin decreased paracellular leakage and increased the tight junctions in glyoxal-treated hRMECs. (a) The FITC-dextran leakage was detected in glyoxal-treated hRMECs with or without melatonin ( n = 3 per group). Western blot analysis of occludin (b), ZO-1 (c), and JAM-A (d) in mean +- SEM ( n = 9-10 per group); p values by one-way ANOVA. * p < .05; ** p < .01; *** p < .001 compared with glyoxal group. (e) Representative images of ZO-1 (green) and claudin-5 (red) immunostainings in glyoxal-treated hRMECs with or without melatonin. The nuclei were counterstained with DAPI (blue). Scale bar = 5 mum. FITC-dextran is representative of three independent experiments. DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; hRMECs, human retinal microvascular endothelial cells

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Experimental details: Figure 1. H. pylori disrupts the tight junction protein JAM-A in gastric epithelial cell lines, primary cells, and gastric biopsy specimens. (A) JAM-A immunofluorescence (green) in uninfected and H. pylori 26695-infected gastric cell lines MKN74, NCI-N87, and AGS-Ecad; scale bar, 10 mum; n = 3. (B) JAM-A immunofluorescence (red) in uninfected and H. pylori 26695-infected human primary gastric epithelial cells. A pancytokeratin (green) antibody was used to confirm the epithelial origin of the cells. Nuclei were counterstained with DAPI (blue); scale bar, 10 mum; n = 3; (C) JAM-A immunohistochemistry in human gastric biopsies specimens of H. pylori -infected and uninfected patients. Normal JAM-A pattern is a continuous staining circumscribing the apical region of epithelial cells (black arrowheads, upper panels). Altered JAM-A is the interruption of the belt pattern, or loss of expression (lower panels). Pictures were originally obtained with a 1000x magnification. (D) Frequency of JAM-A alterations in 52 gastric biopsy specimens according to the H. pylori colonization density (0, absent; 1, mild; 2, moderate; and 3 marked). The two-sided Mann-Whitney U test was used to determine statistical significance

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Experimental details: Figure 2. H. pylori cleaves the cytoplasmic domain of JAM-A. (A) Western blots of JAM-A in uninfected or H. pylori 26695-infected AGS-Ecad cells using antibodies against the cytoplasmic C-terminus and the extracellular N-terminus domain of the protein. Data are representative of n >= 4 experiments; (B) MALDI-TOF mass spectrometry analysis of N-terminus immunoprecipitated JAM-A (from H. pylori 26695-infected and non-infected samples) after separation in SDS-PAGE. Mass spectra were obtained after in gel tryptic digestion of excised bands and peptide peaks related to JAM-A cytoplasmic domains are indicated by arrows with the following correspondence m/z 1020.548 (VIYSQPSAR), m/z 1148.642 (KVIYSQPSAR), and m/z 1458.707 (SEGEFKQTSSFLV). This experiment was performed twice; (C) Protein coverage data from the uninfected and H. pylori -infected samples. Tryptic peptide fragments obtained are depicted in red in the full protein sequence of JAM-A. The JAM-A cytoplasmic domain is identified by the blue boxes. (D and E) MALDI-TOF mass spectrometry analyses of a synthetic peptide of the full cytoplasmic domain of JAM-A incubated with ultra-pure water (D) or with H. pylori 26695 sonicate (E). Results are representative of n >= 5 experiments, each with similar results. Arrows indicate the peak which corresponds to the full peptide (black), and the peaks obtained after incubation with H. pylori (blue, major pair of peptides; red, minor pair of peptides)

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Experimental details: Figure 5. Identification of the H. pylori factor that cleaves JAM-A. (A) Western blot of JAM-A in AGS-Ecad cells infected with H. pylori 60190, 60190CagA - , 60190CagE - , or 60190VacA - mutants, or with clinical isolates using antibodies to detect the C- or N-terminus of JAM-A; tubulin was used as loading control. (B) Screening of the type of protease involved in JAM-A cleavage with a panel of protease inhibitors. Cleavage was evaluated by MALDI-TOF/TOF analysis using a cytoplasmic domain JAM-A peptide ( 277 KVIYSQPSARSEGEFK 292 ) incubated with H. pylori 26695 lysates in the presence of the inhibitors. , cleavage inhibition; X, no cleavage inhibition. Experiments were performed n >= 2, with similar results. c) Upper panel , representative chromatogram obtained after separation of the 100 mM NaCl fraction from Q-Sepharose on SOURCE-15Q. Red boxes, fractions (A2 to A6) selected for size exclusion chromatography (SEC). Middle panel , Coomassie blue-stained SDS-PAGE gel of separated fractions. Bottom panel , detection of the H. pylori protease by western blot after incubation of the fractions with AGS-Ecad cell lysates. H. pylori 26695 and uninfected cell lysates were used as positive and negative controls, respectively. (D) Upper panel , representative chromatogram obtained from SEC after separation of fractions A2 to A6 obtained from SOURCE-15Q. Green box, fractions (A10 to B8) selected for the detection of cleavage activity; Red box, fraction selected for protein identificat

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Experimental details: Figure 3 BmpR1a DeltaFoxL1+ mice displayed abnormalities in goblet cell vesicles and presented dysregulated mucus maturation and structural gene expression. ( A ) Relative mRNA levels of the colonocyte/secretory lineage specification Hes1 and Atoh1 , along with committed-secretory cell toward the goblet fate or maturation Gfi1 , Spdef and Klf4 . A significant decrease was observed for Klf4 mRNA levels in mutant mice while other mRNAs were not modulated between mutant and control mice ( N = 7-9). ( B ) Ultrastructural analysis of the goblet cells revealed vesicles without mucin diversity, disturbed morphology in BmpR1a DeltaFoxL1+ mice ( N = 4). ( C ) Vesicles pattern quantification revealed a significant increase in light grey vesicles in mutant goblet cells when compared to controls ( N = 3). ( D ) BmpR1a DeltaFoxL1+ mice showed a significant increase in Fut2 , Muc2 , Muc4 and Agr2 mRNA levels in mutant mice. ( E ) Ultrastructural analysis of the glycocalyx and the apical junctional complex (yellow bracket) revealed that the latter was not modified between both groups and showed loss of glycocalyx in BmpR1a DeltaFoxL1+ mice ( N = 4). ( F ) Immunofluorescence against ZO-1, claudin-1 and 2 as well as JAM-A revealed no modulation in the tight junction complex proteins between both groups ( N = 3). ( C ) 2-way ANOVA test. Data are presented as the mean +- SD. ( D ) Mann-Whitney test. N: Nucleus; Mu: Mucin vesicles; G: Glycocalyx; AJC: Apical junctional complex. Fold-change was n

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Experimental details: 2 A, B : A study by immunoblotting to assess changes in TJ-associated proteins in the rat testis during androgen suppression-induced germ cell loss and its recovery. A: Immunoblotting results in which lysates of testes containing ~150 mug protein from each sample within an experimental group were resolved by SDS-PAGE, and the blots were probed with different primary antibodies. Immunoblot data were arranged into two groups: androgen depletion induced by TE implants and the two recovery groups. In the recovery phase [either via spontaneous recovery (SR) or under high T condition with 4 x 4 cm T implants (+T4)], the column labeled as 30/35D represents recovery under high T condition on day 30 or spontaneous recovery on day 35. The bottom part is the same blot as those shown above, but reprobed with an anti-actin antibody to assess equal protein loading. B: These are densitometrically-scanned data using immunoblots such as those shown in (A). All data were normalized against beta-actin to account for uneven protein loading and the level of a target protein in control rats was arbitrarily set at 1. Each data point is the mean +- SD of three rats. ns, not significantly different from control as determined by ANOVA; *, significantly different, P < 0.05; **, significantly different, P < 0.01.

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Experimental details: Figure 4 Aroclor1254 accelerated junction protein endocytosis and ubiquitination via p38 MAPK. ( a ) Immunoblot analysis of endocytosed JAM-A, N-cadherin, and beta -catenin in SCs at different time points (0, 10, 30, 60 min) after cell surface biotinylation for 30 min in the presence of Aroclor1254 (or the mixture of Aroclor1254 and SB203580). The endocytosed proteins were pulled-down by UltraLink Immobilized NeutrAvidin Plus Resin after stripping residual biotin on cell membrane as described in 'Materials and methods' section. Total biotinylated proteins at 0 min without stripping were also detected as a positive control. Cell lysates without pull-down were also analyzed by immunoblot to confirm identical levels of the tested protein between groups with GAPDH served as the loading control. ( b ) Line and scatter graphs summarizing the result shown in a by calculating the percentage of endocytosed protein at each data point versus the total biotinylated protein. Each bar refers to mean+-S.D. of n =3 independent experiments using SCs cultured from different rats. * P

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Experimental details: Figure 5 The effects of Aroclor1254 on the barrier function of SC epithelium after inhibiting the caveolin-dependent endocytosis. The SCs were treated with or without Aroclor1254 in the presence or absence of CO as the regimen depicted in Supplementary Figure S2a . ( a and b ) The barrier function of SC epithelium measured by recording TER ( a ) or calculating the permeability of Na-F ( b ) across the cell monolayer. The Na-F permeability in the vehicle control on day 3 was arbitrarily set at 100%. CO treatment was found to partially resist the disruption of BTB by Aroclor1254. Each bar refers to mean+-S.D. of n =3 experiments using different batches of SCs. ** P

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Experimental details: Fig. 5 Re-establishment of BTB in the recipient testes after repaired SSC transplantation. A , B Distribution of N-cadherin and JAM-A (all in green) were found to be different in different tubules, depending on the status of spermatogenesis and the differentiation status of repaired SSCs. Abnormal distribution of these proteins in empty tubules devoid of germ cells (a and g) was obviously noted and gradually returned to normalcy as showed in normal wild type control testis (e and k) following the appearance of spermatocytes (b and h), round spermatids (c and i), and elongated spermatids (d and j). Spermatocytes were labeled by anti-SCP3 antibody (red), and round spermatids and elongated spermatids were identified by the corresponding nucleus shape (blue). There were no germ cells in panels a, a', f, g, g', and l. The boxed areas in a-d and g-j are magnified and shown in a'-d' and g'-j'. Scale bar = 50 mum in a-l and 10 mum in a'-d' and g'-j'