Thymi were harvested and embedded in Tissue-Tek Optimal Cutting Temperature media. Eight micrometer frozen thymic sections were fixed in 100% acetone and blocked in 10% goat serum before incubation with primary antibodies. Primary antibodies were purchased from either Abcam (keratin-5, keratin-8) or eBioscience (Aire) and all secondary antibodies were purchased from Invitrogen. Immunofluorescence slides were visualized using a Zeiss Apotome widefield microscope. For eye disease scoring, eyes were processed by formalin fixation and H&E staining as previously described [6 (link), 8 (link)]. Sections were blindly scored for severity of infiltration and tissue destruction. H&E slides were imaged using a Zeiss AxioImager brightfield microscope.
Axioimager brightfield microscope
Manufactured by Zeiss
The AxioImager brightfield microscope is a high-quality optical instrument designed for various microscopy applications. It features a robust construction, providing a stable platform for precise observation and analysis. The microscope is equipped with advanced optics that deliver clear, high-resolution images, enabling users to examine samples with exceptional clarity. The AxioImager is a versatile tool suitable for a wide range of research and educational purposes.
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Lab products found in correlation
Anti pcna antibody, by Abcam (2 mentions)
Vectastain elite abc rabbit kit, by Vector Laboratories (2 mentions)
Apotome, by Zeiss (2 mentions)
Apotome widefield microscope, by Zeiss (1 mentions)
Ab175385, by Abcam (1 mentions)
Ab9572, by Abcam (1 mentions)
Ab199688, by Abcam (1 mentions)
Apoptag plus in situ apoptosis fluorescein detection kit, by Merck Group (1 mentions)
Scramble control, by Qiagen (1 mentions)
Nbt bcip solution, by Roche (1 mentions)
Anti dig ap antibody, by Roche (1 mentions)
Az100, by Zeiss (1 mentions)
8 protocols using axioimager brightfield microscope
1
Immunofluorescence and Histology of Thymus and Eyes
2
Quantification of Protein Expression in Bone Samples
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A standard indirect IHC procedure was used to detect IGF-1, BMPR1b and MMP-10 expression (Dobie et al., 2015 (link)). Primary antibodies were anti-IGF-1 (Abcam, ab9572, 1:500 dilution), BMPR1b (Abcam, ab175385, 1:100 dilution) and MMP-10 (Abcam, ab199688, 1:100 dilution). Control sections were incubated with an equal amount of rabbit IgG in place of the primary antibody. Sections were counterstained by Haematoxylin, mounted and images captured using a Zeiss AxioImager brightfield microscope. Fixation and antigen retrieval were carried out using the same methodology throughout and all IHC for each antibody was performed on the same day under identical conditions, with control specimens also tested for each genotype. After the images were imported into Fiji, the Haematoxylin-DAB colour deconvolution plugin was used to separate the Haematoxylin and DAB components, and the ‘analyse-measure’ tool used to determine the absorbance in a consistent region of each sample, containing both GP and metaphyseal bone. Optical density (OD) was then calculated using the equation: OD=negative (base10)log of mean intensity of transmitted image/illumination (max intensity of image).
Maximum intensity was taken to be 255 for 8-bit images in Fiji (Ruifrok and Johnston, 2001 (link)).
Maximum intensity was taken to be 255 for 8-bit images in Fiji (Ruifrok and Johnston, 2001 (link)).
3
Apoptosis and Proliferation Analysis in Murine Growth Plate
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The ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit (EMD Millipore, Bedford, MA, USA) was used as per manufacturer's instructions to quantify the number of apoptotic cells in the tibial GP of 7-week-old mice. Proliferating cell nuclear antigen (PCNA) immunohistochemistry (IHC) was performed using a 1:4000 dilution of an anti-PCNA antibody (Abcam, Cambridge, UK) followed by the Vectastain elite ABC rabbit kit (Vector Laboratories, Peterborough, UK) on paraffin-embedded sections from 3-week-old mice (when rates of proliferation were likely to be highest). Sections were counterstained by Haematoxylin and viewed using a Zeiss AxioImager brightfield microscope. The Haematoxylin-DAB colour deconvolution plugin was used in Fiji to calculate the percentage of PCNA+ve cells in the proliferating zone of the GP of each sample.
4
In Situ Hybridization of miR-205 in Thymus
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In situ hybridization was performed on frozen thymic sections. At harvest, thymi were fixed for 2 hours in 4% paraformaldehyde and equilibrated for 7 hours in 30% sucrose. We used double DIG labeled LNA probes against either miR-205 or a scramble control (Exiqon). We followed the manufacturer’s instructions and hybridized the probes overnight at 57°C with the following modifications: Post-hybridization stringency washes: 2x SSC for 60’ at 57°C, 1x SSC for 10’ at RT, 0.5x SSC for 10’ at RT, 0.1x SSC for 45’ at 57°C. Tissues were then blocked with 1% goat serum in 0.1% PBS-Tween-20 (PBST) for 2 hours before overnight incubation with a 1:5000 dilution of anti-DIG-AP antibody (Roche) at 4°C. Following antibody incubation and overnight washes in PBST, alkaline phosphatase activity was detected using an NBT/BCIP solution (Roche). Slides were visualized using either a Zeiss Apotome or a Zeiss AxioImager brightfield microscope.
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In situ hybridization was performed on frozen thymic sections. At harvest, thymi were fixed for 2 hours in 4% paraformaldehyde and equilibrated for 7 hours in 30% sucrose. We used double DIG labeled LNA probes against either miR-205 or a scramble control (Exiqon). We followed the manufacturer’s instructions and hybridized the probes overnight at 57°C with the following modifications: Post-hybridization stringency washes: 2x SSC for 60’ at 57°C, 1x SSC for 10’ at RT, 0.5x SSC for 10’ at RT, 0.1x SSC for 45’ at 57°C. Tissues were then blocked with 1% goat serum in 0.1% PBS-Tween-20 (PBST) for 2 hours before overnight incubation with a 1:5000 dilution of anti-DIG-AP antibody (Roche) at 4°C. Following antibody incubation and overnight washes in PBST, alkaline phosphatase activity was detected using an NBT/BCIP solution (Roche). Slides were visualized using either a Zeiss Apotome or a Zeiss AxioImager brightfield microscope.
5
Histological Analysis of Tibialis Anterior
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The tibialis anterior muscles were removed and fixed in 4% paraformaldehyde before undergoing paraffin wax-embedding. Sections (6 µm) were stained with haematoxylin and eosin for histological assessment, according to the TREAT-NMD protocol (Grounds 2014a ). Images were acquired using a Zeiss AxioImager brightfield microscope and analysed using Fiji (Schindelin et al. 2012 (link)). The percentage of inflammatory cells (characterised by infiltrated dystrophic myofibres with barely visible sarcoplasm) in the ROI was calculated, and the number of central nuclei (signifying muscle regeneration) was recorded, to obtain a cumulative measure of skeletal muscle damage (Grounds 2014a ).
6
Visualization of Fe and Callose in Roots
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Accumulation and distribution of Fe and callose in roots was monitored as previously described [19 (link)]. De-methyl esterified pectins were stained for 5–10 min in 0.05 % (w/v) Ruthenium Red solution (Applichem). Hydroxylamine-ferric chloride staining was adapted from Hornatowska and Reeve [63 (link), 64 ]. Seedlings were initially incubated for 5–10 min in freshly prepared hydroxylamine solution (0.7 % NaOH, 0.7 % hydroxylamine hydrochloride in 60 % EtOH), followed by the addition of an equal (or higher) volume of a solution containing concentrated HCl/EtOH 95 % (1:2 ratio). The solution was removed and ferric chloride was added (10 % FeCl3 in 60 % EtOH containing 0.1 N HCl). Seedlings were cleared using chloral hydrate solution (7:7:1 chloral hydrate:ddH2O:glycerol). Samples were analyzed using a multizoom stereomicroscope (Nikon AZ100) for overview images and a Zeiss AxioImager bright field microscope for detail images.
7
Histological Assessment of Muscle Damage
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The tibialis anterior muscles were removed and fixed in 4% paraformaldehyde (PFA) before undergoing paraffin wax embedding. Sections (6 µm) were stained with Haematoxylin and Eosin (H&E) for histological assessment, according to the TREAT-NMD protocol (Grounds, 2014 ). Images were acquired using a Zeiss AxioImager brightfield microscope and analysed using Fiji (Schindelin et al., 2012 (link)). The percentage of inflammatory cells in the region of interest (ROI) was calculated and the number of central nuclei (signifying muscle regeneration) recorded, to obtain a cumulative measure of skeletal muscle damage.
8
Quantifying Proliferating Cells in Bone
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Proliferating cell nuclear antigen (PCNA) immunohistochemistry (IHC) was performed using a 1:4000 dilution of an anti-PCNA antibody (Abcam) followed by the Vectastain elite ABC rabbit kit (Vector Labs, Peterborough, UK) on paraffin-embedded sections of proximal right tibiae. Sections were counterstained by haematoxylin and viewed using a Zeiss AxioImager brightfield microscope. The haematoxylin–DAB colour deconvolution plugin was used within Fiji to calculate the percentage of PCNA +ve cells in the proliferating zone of the GP of each sample (Schindelin et al. 2012 (link)).
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