Journal: Oncotarget

Article Title: RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma

doi: 10.18632/oncotarget.24079

Figure Lengend Snippet: ( A ) Hepatocellular carcinoma (HCC) tissues and the adjacent non-tumor tissues (NC) from HCC patients were subjected to immunohistochemistry staining with anti-AUF1 and anti-Dicer1 antibodies (×400). ( B ) Total RNA was extracted from HCC tissues and adjacent non-tumor tissues. The relative levels of DICER1 mRNA, AUF1 mRNA, and miR-122 were determined by qRT-PCR compared to GAPDH mRNA. Data are represented as mean ± SD. n = 20; * P < 0.05. ( C ) HL7702, Huh7, and PLC/PRF/5 cells were cultured in 6-well plate to 80% confluency. Cellular proteins were extracted, and the expression of Dicer1 and AUF1 was determined by Western blotting. AUF1 includes four isoforms (37, 40, 42, and 45 kDa). ( D) HHL-5 and Huh7 cells were cultured in 6-well plate to 80% confluency. The level of DICER1 mRNA and AUF1 mRNA was determined by qRT-PCR normalized to GAPDH mRNA. Data are represented as mean ± SD. n = 4, ** P < 0.01.

Article Snippet: Briefly, the 3′UTR sequence of DICER1 mRNA was amplified by PCR from plasmid pIS1 DICER1 long UTR (Addgene, Cambridge, MA) with the forward primer 5′-ATATATGGATCCAACCGCTTTTTAAAATTCAAAACAA-3′ and reverse primer 5′-GCGCTAGGGCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTGAACAGACGAT-3′.

Techniques: Immunohistochemistry, Staining, Quantitative RT-PCR, Cell Culture, Expressing, Western Blot

Journal: Oncotarget

Article Title: RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma

doi: 10.18632/oncotarget.24079

Figure Lengend Snippet: ( A , C ) PLC/PRF/5 (A) and Huh7 cells (C) were grown in 6-well plate to 70% confluency. Cells were transfected with pEGFP-AUF1 (AUF1) or siRNA of AUF1 (siAUF1) for 48 h. Control cells were transfected with liposome (NC), pEGFP-C1 (C1), and mock siRNA (siMock), respectively. Proteins were extracted and subjected to Western blot analysis. ( B , D) The level of DICER1 mRNA was determined by qRT-PCR. Data are represented as mean ± SD. n = 4; ** P < 0.01. Experiments were repeated three times and representative results were presented.

Article Snippet: Briefly, the 3′UTR sequence of DICER1 mRNA was amplified by PCR from plasmid pIS1 DICER1 long UTR (Addgene, Cambridge, MA) with the forward primer 5′-ATATATGGATCCAACCGCTTTTTAAAATTCAAAACAA-3′ and reverse primer 5′-GCGCTAGGGCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTGAACAGACGAT-3′.

Techniques: Transfection, Control, Western Blot, Quantitative RT-PCR

Journal: Oncotarget

Article Title: RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma

doi: 10.18632/oncotarget.24079

Figure Lengend Snippet: ( A ) The diagram of pEGFP-Dicer1-3′UTR. ( B ) Vero cells were co-transfected with pEGFP-Dicer1-3′UTR and pmCherry-AUF1 for 24 h, and the expression of EGFP and AUF1 was observed by fluorescence microscopy. ( C ) The fluorescence quantity was measured by fluorospectrometer. ( D ) The expression of EGFP protein in the treated Vero cells. n = 3. ** P < 0.01. Experiments were repeated three times and representative results were presented.

Article Snippet: Briefly, the 3′UTR sequence of DICER1 mRNA was amplified by PCR from plasmid pIS1 DICER1 long UTR (Addgene, Cambridge, MA) with the forward primer 5′-ATATATGGATCCAACCGCTTTTTAAAATTCAAAACAA-3′ and reverse primer 5′-GCGCTAGGGCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTGAACAGACGAT-3′.

Techniques: Transfection, Expressing, Fluorescence, Microscopy

Journal: Oncotarget

Article Title: RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma

doi: 10.18632/oncotarget.24079

Figure Lengend Snippet: ( A ) The diagram of pEGFP-Dicer1-ORF. ( B ) Vero cells were co-transfected with pEGFP-Dicer1-ORF and pmCherry-AUF1 for 24 h, and the expression of EGFP and AUF1 was observed by fluorescence microscopy. ( C ) The fluorescence quantity was measured by fluorospectrometer. ( D ) EGFP protein expression in the treated Vero cells. n = 3. ** P < 0.01. Experiment was repeated three times and representative results were presented.

Article Snippet: Briefly, the 3′UTR sequence of DICER1 mRNA was amplified by PCR from plasmid pIS1 DICER1 long UTR (Addgene, Cambridge, MA) with the forward primer 5′-ATATATGGATCCAACCGCTTTTTAAAATTCAAAACAA-3′ and reverse primer 5′-GCGCTAGGGCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTGAACAGACGAT-3′.

Techniques: Transfection, Expressing, Fluorescence, Microscopy

Journal: Oncotarget

Article Title: RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma

doi: 10.18632/oncotarget.24079

Figure Lengend Snippet: Huh7 cells were transfected with siAUF1 and siDicer1 for 36–48 h. ( A – E ) The levels of oncogenic miRNAs (miR-1, miR-21, miR-125b, miR-375) and miR-122 were determined by qRT-PCR. ( F ) The mRNAs of AUF1 and DICER1 were detected by qRT-PCR at 36 h post-transfection to show the knockdown efficiency of siRNAs. Control cells were transfected with siMock. The abundance of miRNAs and mRNAs was normalized to GAPDH mRNA. Data are presented as mean ± SD. n = 4. ** P < 0.01.

Article Snippet: Briefly, the 3′UTR sequence of DICER1 mRNA was amplified by PCR from plasmid pIS1 DICER1 long UTR (Addgene, Cambridge, MA) with the forward primer 5′-ATATATGGATCCAACCGCTTTTTAAAATTCAAAACAA-3′ and reverse primer 5′-GCGCTAGGGCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTGAACAGACGAT-3′.

Techniques: Transfection, Quantitative RT-PCR, Knockdown, Control

Journal: Oncotarget

Article Title: RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma

doi: 10.18632/oncotarget.24079

Figure Lengend Snippet: ( A ) PLC/PRF/5 cells were grown in 6-well plate. The cells were transfected with pmCherry-AUF1 (AUF1) or siRNA of AUF1 (siAUF1) for 24 h in the medium without serum. Apoptosis was determined by flow cytometry. Control cells were transfected with liposome (NC), pmCherry-C1 (C1) or siMock. ( B ) 80% confluent HHL-5 cells were transfected with siAUF1 or siMock for 24 h in the medium without serum. The expression of Dicer1, AUF1, and cleaved PARP1 was determined by Western blotting (a). The relative expression levels are presented as mean ± SD (b, c, d). n = 4, * P < 0.05, ** P < 0.01.

Article Snippet: Briefly, the 3′UTR sequence of DICER1 mRNA was amplified by PCR from plasmid pIS1 DICER1 long UTR (Addgene, Cambridge, MA) with the forward primer 5′-ATATATGGATCCAACCGCTTTTTAAAATTCAAAACAA-3′ and reverse primer 5′-GCGCTAGGGCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTGAACAGACGAT-3′.

Techniques: Transfection, Flow Cytometry, Control, Expressing, Western Blot

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: MiRNA-based short-hairpins (miR5 and miR7) targeting distinct regions of ATP10B mRNA, and a scrambled non-targeting sequence (SCR) packaged in AAV2/7 vector under a neuronal promoter (CMVenhanced-synapsin) were stereotactically injected in the right SNpc of rats. (A) Overview of the protocol followed in this study and vector constructs. (B, C) The percentage of double TH+ RFP+ cells within the total TH+ population in the SNpc was determined for the three vectors 2 weeks post-injection. (B) Each data point represents an individual animal, and the number of TH+ and TH+ RFP+ cells from 8 sections was quantified using stereological methods. (C) Representative image of the SNpc of a rat injected with the miR5 vector 2 weeks post-injection, fluorescently stained with TH and RFP antibodies, shows efficient transduction of dopaminergic neurons.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Sequencing, Plasmid Preparation, Injection, Construct, Staining, Transduction

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: (A, C, G) Indicative of decreased ipsilateral dopaminergic neurotransmission, rats with ATP10B KD showed decreased preference in the use of the contralateral paw in the cylinder test (A), increased bias to ipsilateral swing in the elevated body swing test (C), and spontaneous ipsilateral turning in the open field test (G). (B) Motor coordination and balance was assessed using an accelerated rotarod test (4-40 rpm), with the average performance post-lesion normalized to the individual rat performance prior to surgery. (D, E, F) Spontaneous motor behavior was recorded during a 5-min open field test and analyzed for total distance traveled (D), velocity (E) and rearing (F). Data are mean + s.e.m. *** p < 0.001 (two-way ANOVA, treatment factor), # p < 0.05, ## p < 0.01, ### p < 0.001 (Dunnett’s post hoc test miR5 vs. SCR at corresponding time point), $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 (Dunnett’s post hoc test miR7 vs. SCR at corresponding time point).

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques:

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: ATP10B KD in SNpc neurons leads to Parkinsonian motor deficits at 12 months post injection . (A) Motor coordination and balance was assessed using an accelerated rotarod test (4-40 rpm), with the average performance post-lesion normalized to the individual rat performance prior to surgery (p = 0.0083; SCR vs. miR5 p = 0.0043, SCR vs. miR7 p = 0.1111). (B, C, H) Motor asymmetry was examined by quantifying the preference in the use of the contralateral paw in the cylinder test (p = 0.0110; SCR vs. miR5 p = 0. 00068, SCR vs. miR7 p = 0. 0799), the bias to ipsilateral swing in the EBST (p = 0.0016; SCR vs. miR5 p = 0.0321, SCR vs. miR7 p = 0.0009), and the spontaneous ipsilateral turning behavior in the open field test (p = 0.0123; SCR vs. miR5 p = 0.0388, SCR vs. miR7 p = 0.0112). (E, F, G) Spontaneous motor behavior was recorded during a 5-min open field test and analyzed for total distance traveled (p = 0.0380; SCR vs. miR5 p = 0.1035, SCR vs. miR7 p = 0.0312), velocity (p = 0.0249; SCR vs. miR5 p = 0.0722, SCR vs. miR7 p = 0.0212), and frequency of rearing (p = 0.0064; SCR vs. miR5 p = 0.0083, SCR vs. miR7 p = 0.0166). (D) The catalepsy bar test was performed to assess muscular rigidity. Time spent by the rat grabbing the elevated bar before correcting its posture on the floor was measured (p = 0.0174; SCR vs. miR5 p = 0.0181, SCR vs. miR7 p = 0.0481). In (A, C-H) data are mean ± s.e.m, and analyzed using non-parametric one-way ANOVA (Kruskall Wallis) and Dunn’s post-hoc test versus SCR. SCR (n=15), miR5 (n=15), miR7 (n=17). In (B) data are mean ± s.e.m and analyzed using one-way ANOVA and Dunnet’s post-hoc test versus SCR. SCR (n=12), miR5 (n=10), miR7 (n=11). Each dot represents the value of one animal.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Injection

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: Striatal DAT binding potential (BPnd), measured by 18F-FE-PE2I microPET imaging, in the ipsilateral (right) and the contralateral (left) striatum observed in miR5-injected animals (n=5), SCR-injected animals (n=3) and miR7-injected animals at 7-9 months (n=2) and 12 months (n=3).

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Binding Assay, Imaging, Injection

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: (A) Striatal DAT binding potential (BPnd), measured by 18 F-FE-PE2I microPET imaging, in the ipsilateral (R) vs. the contralateral (L) striatum in miR5-injected animals (n=5) and SCR-injected animals (n=3) (treatment factor p = 0.0007; SCR vs. miR5 2-4 months p = 0.0134, SCR vs. miR5 4-6 months p = 0.0448, SCR vs. miR5 7-9 months p = 0.0448, SCR vs. miR5 12 months p = 0.0024). Data are mean ± s.e.m. and analyzed using two-way ANOVA and Sidak’s post hoc test miR5 vs. SCR at corresponding time point. The effect could be reproduced in miR7-injected animals at 7-9 months (n=2) and 12 months (n=3). (B) Parametric images of 18 F-FE-PE2I BPnd in representative SCR (upper) and miR5 (middle) rats over time; lower row represents representative miR7 images at the final two time points. Transverse images are shown in neurological orientation and are overlaid onto a reference CT skull template with the miR5/miR7 lesioned striatum on the right-hand side.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Binding Assay, Imaging, Injection

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: ATP10B KD in SNpc neurons leads to time-dependent loss of dopaminergic terminals in the dSTR . TH positive area was quantified using QuPath in 8 different sections that cover the STR. (A) TH positive area in the contralateral (Contra) and ipsilateral (Ipsi) dorsal STR of 1 month post-injection rats (SCR Contra vs. SCR Ipsi p = 0.5704, miR Contra vs. miR5 Ipsi p = 0.0046, miR7 Contra vs. miR7 Ipsi p = 0.6951). SCR (n=6), miR5 (n=6), miR7 (n=6). (B) Representative image of TH immunohistochemical staining on 4 different sections from one SCR, miR5 and miR7 rat 1 month post-injection. (C) TH positive area in the contralateral and ipsilateral dorsal STR of 1 year post-injection rats(SCR Contra vs. SCR Ipsi p = 0.5397, miR Contra vs. miR5 Ipsi p = 0.0025, miR7 Contra vs. miR7 Ipsi p = 0.0026). SCR (n=8), miR5 (n=8), miR7 (n=8). (D) Representative image of TH immunohistochemical staining on 4 different sections from one SCR, miR5 and miR7 rat 1 year post-injection. Data are mean ± s.e.m, and anayzed using a paired t-test. Each dot represents the average of the 8 sections per animal.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Injection, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: TH positive area was quantified using ImageJ in 6 different sections that cover the STR. (A) Percentage of TH positive area ipsilateral (R) versus contralateral (L) dorsal STR of 1 year post-injection rats, SCR (n=5) and miR5 (n=7). Data are mean ± s.e.m and analyzed using t test, ** p = 0.0025. Each dot represents the average of the 6 sections per animal. (B) Representative image of TH immunohistochemical staining on 6 different sections from one SCR and miR5 rat 1 year post-injection.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Injection, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: TH positive cells were counted using stereology in 7 different sections that cover the SNpc. (A) Number of TH+ cells in the contralateral (Contra) and ipsilateral (Ipsi) SNpc of 1 month post-injection rats(SCR Contra vs. SCR Ipsi p = 0.98, miR Contra vs. miR5 Ipsi p = 0.2227, miR7 Contra vs. miR7 Ipsi p = 0.0160). SCR (n=6), miR5 (n=6), miR7 (n=5). (B) Representative image of TH immunofluorescence staining on 1 SNpc section from one SCR, miR5 and miR7 rat 1 month post-injection. (C) Number of TH+ cells in the contralateral (Non Inj) and ipsilateral (Inj) SNpc of 1 year post-injection rats(SCR Contra vs. SCR Ipsi p = 0.7125, miR Contra vs. miR5 Ipsi p = 0.0071, miR7 Contra vs. miR7 Ipsi p < 0.0001). SCR (n=8), miR5 (n=8), miR7 (n=8). (D) Representative image of TH immunofluorescence staining on 1 SNpc section from one SCR, miR5 and miR7 rat 1 year post-injection. Data are mean ± s.e.m, and analyzed using a paired t-test. Each dot represents the average of the 7 sections per animal.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Injection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: (A-E) Protein signal values were normalized to the endogenous protein (β-actin) levels, and the ipsilateral (Ipsi) versus contralateral (Contra) percentage of the protein signal is represented. Data are presented as mean ± s.e.m., with each dot representing an individual animal. (F) Representative western blot images of α-synuclein and lysosomal proteins analyzed in whole SN extracts from SCR, miR5, and miR7 rats.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: (A) Representative immunofluorescent images of LAMP1, LAMP2a, cathepsin B and Gcase co-stained with TH marker in the SNpc of SCR, miR5 and miR7 rats at 1 year post-injection. (B-E) Density of LAMP1+, LAMP2a+, cathepsin B+, or Gcase+ spots (number of spots per total TH+ surface) and average spot volume in TH+ cells quantified in the injected side versus non-injected side. SCR (n=8), miR5 (n=8), miR7 (n=8). (B) LAMP1+ number of spots (SCR vs. miR5 p = 0.9089, SCR vs. miR7 p < 0.0001) and LAMP1+ average spot volume (SCR vs. miR5 p > 0.9999, SCR vs. miR7 p = 0.0060). (C) LAMP2a+ number of spots (SCR vs. miR5 p = 0.0017, SCR vs. miR7 p < 0.0001) and LAMP2a+ average spot volume (SCR vs. miR5 p = 0.9435, SCR vs. miR7 p = 0.0009). (D) Cathepsin B+ number of spots (SCR vs. miR5 p = 0.0016, SCR vs. miR7 p < 0.0001) and cathepsin B+ average spot volume (SCR vs. miR5 p = 0.5022, SCR vs. miR7 p < 0.0001). (E) Gcase+ number of spots (SCR vs. miR5 p = 0.9987, SCR vs. miR7 p = 0.568) and Gcase+ average spot volume (SCR vs. miR5 p = 0.0012, SCR vs. miR7 p = 0.0020). Data are mean ± s.e.m and analyzed using one-way ANOVA followed Šídák’s multiple comparisons test, with the exception of LAMP1 average spot volume which was analyzed using Kruskal-Wallis followed Dunn’s multiple comparisons test. Each dot represents the average of 3 sections per animal.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Staining, Marker, Injection