Journal: Physiological Reports

Article Title: Tuberous sclerosis complex exhibits a new renal cystogenic mechanism

doi: 10.14814/phy2.13983

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Rabbit polyclonal Anti‐NCC , 1:60 , Stressmarq Biosciences; Victoria, BC, Canada , SPC‐402.

Techniques: Plasmid Preparation, Generated

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Friedreich Ataxia: Developmental Failure of the Dorsal Root Entry Zone

doi: 10.1093/jnen/nlx087

Figure Lengend Snippet: DREZ in a normal control and a patient with FA shown by α-dystroglycan immunohistochemistry and double-label immunofluorescence of periaxin and GFAP. (A, B) Normal control; FA (C, D) (FA2, Table, GAA 1200/1200). (A) In the normal control, compact α-dystroglycan reaction product surrounds the dome of the CNS portion of the DREZ. (B) Double-label immunofluorescence of periaxin and GFAP shows a relatively sharp transition of myelinating Schwann cells (red) to GFAP-reactive CNS tissue (green) in the DREZ. (C) In FA, the demarcation between α-dystroglycan-positive and negative regions of the DREZ is interrupted by short breaks where CNS tissue penetrates into the DR (arrows). (D) In FA, the normal demarcation between Schwann cells (periaxin) and CNS (GFAP) is indistinct. (A, C) α-dystroglycan; (B, D) double-label immunofluorescence of periaxin (red) and GFAP (green). Scale bars: (A, C) 200 µm; (B, D) 100 µm.

Article Snippet: Sources of commercial antibodies; catalog numbers; host species and clonality; final protein concentrations or, in case of ascites fluid, dilution; and antigen retrieval methods were as follows: anti-PMP-22 (Abcam, Cambridge, MA, ab-31851, rabbit polyclonal, 2 μg protein/mL, incubation in 80% ethanol at 4 °C overnight); anti-P0 (Abcam, ab15508, rabbit polyclonal, 1 μg protein/mL, incubation in 80% ethanol at 4 °C overnight, followed by 0.01 M citric acid-sodium citrate buffer, pH 6, for 20 minutes at 95 °C); anti-PLP (Abcam, ab-105784, rabbit polyclonal, 2 μg protein/mL, incubation in 80% ethanol at 4 °C overnight, followed by 0.01 M citric acid-sodium citrate buffer, pH 6, for 20 minutes at 95 °C); anti-α-dystroglycan (Novus Biologicals, Littleton, CO, NBP1-49634, mouse monoclonal, 0.4 μg protein/mL, 0.01 M citric acid-sodium citrate buffer, pH 6, for 20 minutes at 95 °C); anti-periaxin (Novus, NBP1-89598, rabbit polyclonal, 2 μg protein/mL, 0.01 M citric acid-sodium citrate buffer, pH 6, 20 minutes at 95 °C); anti-laminin (Sigma-Aldrich, St. Louis, MO, L8271, mouse monoclonal, ascites fluid, diluted 1:1000, proteinase K [Sigma, P-6556], 0.1 μg/mL in tris buffer, pH 8, containing 0.01 M NaCl for 30 minutes at 37 °C); anti-GFAP (BioLegend, San Diego, CA, 837201, mouse monoclonal, ascites fluid, diluted 1:500, DIVA [a proprietary decloaking solution sold by Biocare Medical, Pacheco, CA] 1× for 30 minutes at 95 °C); anti-vimentin (Santa Cruz Biotechnology, Santa Cruz, CA, sc-66002, mouse monoclonal, 2 μg protein/mL, 0.01 M citric acid-sodium citrate buffer, pH 6, 20 minutes at 95 °C); anti-nestin (Santa Cruz, sc-23927, mouse monoclonal, 2 μg protein/mL, 0.01 M citric acid-sodium citrate buffer, pH 6, for 20 minutes at 95 °C, or DIVA for 30 minutes at 95 °C, or trypsin [DIFCO Laboratories, Detroit, MI, 0152-13] 1 mg/mL in 0.07 M CaCl 2 at pH 7.8 for 30 minutes at 37 °C); anti-S100 (Santa Cruz, sc-53438, mouse monoclonal, 0.8 μg protein/mL, 0.01 M citric acid-sodium citrate buffer, pH 6, 20 minutes at 95 °C); anti-class-III-β-tubulin (R&D Systems, Minneapolis, MN, MAB1195, mouse monoclonal, 2 μg protein/mL, 0.01 M citric acid-sodium citrate buffer, pH 6, 20 minutes at 95 °C); anti-phosphorylated neurofilament protein (Covance [now BioLegend] SMI-31 R, mouse monoclonal, ascites fluid, diluted 1:500, 0.01 M citric acid-sodium citrate buffer, pH 6, 20 minutes at 95 °C).

Techniques: Immunohistochemistry, Immunofluorescence

Journal:

Article Title: Improved Hepatic Gene Transfer by Using an Adeno-Associated Virus Serotype 5 Vector

doi: 10.1128/JVI.76.20.10497-10502.2002

Figure Lengend Snippet: Estimation of vector gene copy number in AAV-transduced livers by quantitative PCR (3 months after vector administration). A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems. Template for PCR was as follows: 100 ng of genomic DNA extracted from several random pieces of liver and subsequently combined for each individual animal. Each sample column represents an individual animal. standards, linearized plasmid pAAV-EF1α-hF.IX (0.01, 0.1, or 1 pg) mixed with 100 ng of genomic mouse DNA (extracted from untransduced animal); NC, negative control (template, genomic DNA from untransduced animal). Bands were analyzed by densitometric scanning, and intensities were quantitated with NIH Image 6.16 software. Shown is one representative blot. Ratios of band intensities (hF.IX band/mAAT band) are for the blot shown. Gene copy number estimates are average for two experiments.

Article Snippet: A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems.

Techniques: Plasmid Preparation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control, Software