6g10 2c7  (Developmental Studies Hybridoma Bank)

Journal: Development (Cambridge, England)

Article Title: Planarian microtubules form a network within muscle and regulate injury-induced genes essential for regeneration patterning

doi: 10.1242/dev.204669

Figure Lengend Snippet: Planarian TUBA-2 marks muscle microtubules. (A-C) Planarians were Carnoy's fixed and stained using anti-TUBA-2 antibody and/or 6G10 muscle antibody using (A) 10× (max projection), (B) 20× (max projection), (C) 40× ( z -slices) objectives, showing detection on filaments running along each 6G10 + fiber. 6G10 preferentially stains circular and diagonal fibers; longitudinal fiber images show an additionally brightened 6G10 signal for visualization. (D) Carnoy's fixative enabled only weak detection of nuclei by Hoechst staining, but in rare cases candidate cell bodies of TUBA-2-stained muscle fibers could be identified. (E) Immunostaining TUBA-2 and FISH of collagen + muscle cell bodies (NAFA fix) . Image on the right is a magnified view of the image on the left. (F,G) Colchicine or nocodazole caused a reduction to TUBA-2 muscle fiber staining (magenta arrows) and signal accumulation in hypothesized muscle cell bodies (blue arrows). Samples were Carnoy's fixed and imaged at 20× (except 400 ng/ml nocodazole, imaged at 40×). (G) Bottom panels are higher-magnification views of the top panels. Scorings indicate the number of animals similar to representative images. Detection of TUBA-2 used fluorescent secondary antibodies (A-D,G) or tyramide amplification (E,F). Scale bars: 300 μm (G, top panels); 50 μm (A,B,D-F, bottom panels in G); 25 μm (C).

Article Snippet: For immunostaining with anti-TUBA-2 (this study) and 6G10 (Developmental Studies Hybridoma Bank 6G10-2C7) antibodies, animals were blocked with 10% horse serum (Sigma H1138-500ML) and 10% Roche Western Blocking Reagent (Roche 11921673001) in PBSTx then incubated overnight at room temperature with primary antibody (anti-TUBA-2 polyclonal rabbit antibody at 1:1000, 6G10 mouse monoclonal antibody at 1:1000) in blocking solution.

Techniques: Staining, Immunostaining, Amplification

Journal: Development (Cambridge, England)

Article Title: Planarian microtubules form a network within muscle and regulate injury-induced genes essential for regeneration patterning

doi: 10.1242/dev.204669

Figure Lengend Snippet: Muscle microtubules undergo dynamic changes during wound repair. (A,B) Co-immunostaining with (A) anti-TUBA-2 and (B) 6G10 after incisions in animals treated for 24 h with 125 µg/ml colchicine and recovery in drug-free media. In controls, TUBA-2 + projections extended from wound sites by 4 h (green arrows), wounds appeared closed by 6 h and elevated TUBA-2 levels on microtubules crossing the wound site were detected at 18-24 h (orange arrows). 125 µg/ml colchicine treatment resulted in reduced TUBA-2 staining at wound sites, a lack of TUBA-2 + fibers protruding from the wound edges by 4 h (magenta arrows) and a reduction in fibers along with cell body expression by later timepoints (18-24 h). Animals treated with 125 µg/ml colchicine all healed their wounds by ∼6 h. Muscle fibers protruding from wound edge at 4 h and crossing the wound site at 18 h were 6G10 + . Colchicine-treated animals lacked 6G10 + fiber protrusions extending across the wound site at these times (magenta arrows), although staining was present at reduced levels away from the wound site. (C) Animals were exposed to 6000 Rads of X-rays 3 days before incision and immunostaining for anti-TUBA-2/6G10, and compared to controls. All animals succeeded in healing the TUBA-2/6G10 fiber network across the wound. Scorings show the number of animals similar to representative images. TUBA-2 was detected by tyramide amplification and 6G10 by fluorescent secondary antibodies. Scale bars: 50 μm.

Article Snippet: For immunostaining with anti-TUBA-2 (this study) and 6G10 (Developmental Studies Hybridoma Bank 6G10-2C7) antibodies, animals were blocked with 10% horse serum (Sigma H1138-500ML) and 10% Roche Western Blocking Reagent (Roche 11921673001) in PBSTx then incubated overnight at room temperature with primary antibody (anti-TUBA-2 polyclonal rabbit antibody at 1:1000, 6G10 mouse monoclonal antibody at 1:1000) in blocking solution.

Techniques: Immunostaining, Staining, Expressing, Amplification