Journal: Journal of molecular histology

Article Title: TOB1 and TOB2 mark distinct RNA processing granules in differentiating lens fiber cells.

doi: 10.1007/s10735-023-10177-y

Figure Lengend Snippet: Fig. 1 TOB mRNA expression in developing lens. a. RT-PCR anal ysis showing presence of Tob1 and Tob2 transcripts in RNA iso lated from E15.5, E17.5 and P3 lenses. P3 lenses were separated into epithelial (Epi) and fibers (Fib) prior to RNA extraction. The presence (+) or absence (-) of reverse transcriptase in reac tions is as indicated. M indicates the lane with the 100 bp marker. b. In situ hybridization for Tob1 and Tob2 in eye sections from E15.5 embryos. Intense expres sion of Tob1 was detected in the fiber cells of the transitional zone (tz), with weaker signal in the anterior epithelium. Overall signal for Tob2 was weaker, with distinct signal detected in the anterior epithelium (e) and transitional zones (tz). For both probes the signal was low in the germinative zones (gz) and absent in differentiated fibers (f). Scale bar, 100 μm

Article Snippet: Sections were incubated for 30 min at room temperature (RT) in blocking buffer (3% horse serum in PBS supplemented with 0.1% BSA) prior to incubation overnight with primary antibodies to TOB1 (1:200, mouse monoclonal, SC-133,095, Santa Cruz Biotechnology, Dallas, TX, USA), TOB2 (1:200, goat polyclonal, SC-18,551, Santa Cruz), DCP2 (1:200, rabbit polyclonal, ab28658, AbCam, Cambridge, UK), E-cadherin (1:200, mouse monoclonal, 610,182, BD Biosciences, Mulgrave VIC, Australia), phospho histone-3 (1:200, 07-424, Millipore, Bayswater VIC, Australia), EIF3B (1:100, A301-761 A, Bethyl Labs/Fortis Life Sciences, Cannon Hill QLD, Australia), and β-catenin (1:200, 610,153, BD Biosciences).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, RNA Extraction, Reverse Transcription, Marker, In Situ Hybridization

Journal: Journal of molecular histology

Article Title: TOB1 and TOB2 mark distinct RNA processing granules in differentiating lens fiber cells.

doi: 10.1007/s10735-023-10177-y

Figure Lengend Snippet: Fig. 3 TOB2 is localized in lens fiber cells but does not appear to co-localize with DCP2. Localiza tion of TOB2 (a, c, e) and DCP2 in adjacent paraffin sections (b, d, f) of E13.5 (a, b), E14.5 (c, d) and E16.5 (e, f) lenses. At all ages there is distinct granular or vesicular staining for TOB2 in the transitional zone, which increases in the more differenti ated central fibers (f), with weak staining present in the germina tive zone epithelium (gz) and little or no staining in the anterior epithelium (e). The granules are often arranged in distinct linear arrays along the long axis of the fiber cells; most evident near fiber cell nuclei (inset e, arrowheads). b-c. DCP2 was localized in adjacent sections and while also detected in fiber cells, it shows a distinctly different pattern, labelling large cytoplas mic granules (d, arrowheads). f. Pre-incubating the TOB2 antibody with TOB2 peptide blocked all reactivity. Scale bar: A, B, 100 μm; c, d, 40 μm; e, f, 80 μm; inset, 25 μm. Dashed line indicates lens equator in a, c, e

Article Snippet: Sections were incubated for 30 min at room temperature (RT) in blocking buffer (3% horse serum in PBS supplemented with 0.1% BSA) prior to incubation overnight with primary antibodies to TOB1 (1:200, mouse monoclonal, SC-133,095, Santa Cruz Biotechnology, Dallas, TX, USA), TOB2 (1:200, goat polyclonal, SC-18,551, Santa Cruz), DCP2 (1:200, rabbit polyclonal, ab28658, AbCam, Cambridge, UK), E-cadherin (1:200, mouse monoclonal, 610,182, BD Biosciences, Mulgrave VIC, Australia), phospho histone-3 (1:200, 07-424, Millipore, Bayswater VIC, Australia), EIF3B (1:100, A301-761 A, Bethyl Labs/Fortis Life Sciences, Cannon Hill QLD, Australia), and β-catenin (1:200, 610,153, BD Biosciences).

Techniques: Staining