WT zebrafish eyes (~6 mpf) were enucleated and fixed in 4% paraformaldehyde/PBS at 4 °C overnight. After washing 3 times in PBS for 10 minutes, the eyes were incubated in 10%, 20% and 30% sucrose/PBS at 4 °C overnight each time. The eyes were frozen embedded in Tissue-Tek O.C.T embedding medium (VWR) using dry ice and 14 um sections were collected onto Superfrost PLUS slides (VWR) using a Leica CM1850 cryostat. Tissue was washed with 1X PBS for 5 minutes to remove O.C.T, followed by boiling in RNAscope Target Retrieval reagent (Advanced Cell Diagnostics, ACD) for 5 minutes. Afterwards, slides were briefly washed with sterile water and incubated for 15 minutes at 40 °C with RNAscope Protease III reagent (ACD). Fluorescent in situ hybridization staining was performed using the RNAscope Fluorescent Multiplex Detection kit (ACD) according to the user’s manual. The kcnj13 target probe and odc1 and dapB control probes were designed and provided by ACD. Slides were mounted in Prolong Gold Antifade mountant (Thermo Fisher) and imaged using a Leica LSM 710 confocal microscope.
Oct embedding medium
Manufactured by Avantor
Sourced in France
The OCT-embedding medium is a laboratory product used for the preparation of tissue samples for optical coherence tomography (OCT) analysis. It provides a stable matrix to embed and support the tissue during the sectioning process. The medium ensures the preservation of the tissue structure, allowing for accurate imaging and analysis.
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Lab products found in correlation
Axio imager m2 microscope, by Zeiss (2 mentions)
Superfrost plus slides, by Avantor (1 mentions)
Lsm 710 confocal microscope, by Leica (1 mentions)
Cm1850 cryostat, by Leica (1 mentions)
Rnascope target retrieval reagent, by Advanced Cell Diagnostics (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Mp 7401, by Vector Laboratories (1 mentions)
Superfrost ultra plus slides, by Thermo Fisher Scientific (1 mentions)
Cytospin 4, by Thermo Fisher Scientific (1 mentions)
Anti cd31 antibody, by BD (1 mentions)
Micros 60, by Horiba (1 mentions)
Xe 5000 instrument, by Sysmex (1 mentions)
4 protocols using oct embedding medium
1
Fluorescent in situ Hybridization of Zebrafish Eye
2
Tissue Harvesting and Preservation
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Rats were killed by anaesthetic overdose and the L4/5 DRG, plantar surface of hindpaws and sciatic nerves were rapidly dissected and either snap frozen on dry ice, or post-fixed for 4 h in 4% paraformaldehyde (in 0.1 M phosphate buffer (PB)), cryoprotected (10–30% sucrose in 0.1 M PB at 4 °C over 48 h), frozen in OCT-embedding medium (VWR, UK) and then stored at − 80 °C.
3
Immunohistochemistry of Tumor Tissues
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Excised tumor tissues (U-87 MG, PANC-1, CAL-33) were frozen in OCT-embedding medium (VWR international, Fontenaysous-Bois, France), cut into 7 µm thick sections using a cryomicrotome and mounted onto SuperFrost Ultra Plus slides (Thermo Fisher Scientific). The sections were then fixed with 4% formalin, subjected to heat-mediated antigen retrieval, blocked with BLOXALL (SP600, Vector laboratories) and 2.5% horse serum (MP 7401, Vector laboratories), incubated with primary antibodies (anti-LDLR antibody [EP1553Y] (ab52818) or SR-BI antibody [EPR20190] (ab217318)) overnight at 4 °C, incubated with secondary antibody ImmPRESS™ HRP Polymer Anti-Rabbit IgG for 30 min at RT, and revealed using ImmPACT™ NovaRED™ Diluent (SK 4805, Vector laboratories). To localize the nucleus, the slides were counterstained with Hematoxylin. Finally, the slides were dehydrated using ethanol and xylene, mounted with merckoglass and cover slips. The sections were imaged using Zeiss AxioImager M2 microscope.
4
Multimodal Analysis of Hematopoietic and Tumor Samples
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Blood samples were evaluated for hematological parameters with a medical automated hematology analyzer (Micros-60, Horiba ABX, Montpellier, France), and biochemical analyses were performed by the Charles Rivers Laboratory (Massachusetts, USA). Bone marrow was obtained after having carefully sectioned mice femurs at each joint and flushing the bone cavity with PBS. Cells from the plasma and bone marrow samples were washed in PBS, cytospun (Cytospin 4, Fisher Scientific, Illkirch, France) and stained with May-Grunwald-Giemsa solution (MGG) with the automated XE-5000 Instrument (Sysmex, Roissy, France). Global cell abundance evaluation and megakaryocyte and leukocyte counting were then performed for both samples by an experienced hematologist.
Tumor tissues were frozen in OCT-embedding medium (VWR international, Fontenay-sous-Bois, France) and cut into 7 μm thick sections using a cryomicrotome for immunohistochemical analyses. Immunohistochemical examinations were performed after fixation with 4% paraformaldehyde and staining with anti-CD31 antibody (Pharmingen, BD Biosciences, Le Pont de Claix, France), using an AxioImager M2 microscope (Carl Zeiss, Jena, Germany). Segmentation of tissues was performed using a fully connected, three-layer back-propagation neuronal network, as previously described [12] .
Tumor tissues were frozen in OCT-embedding medium (VWR international, Fontenay-sous-Bois, France) and cut into 7 μm thick sections using a cryomicrotome for immunohistochemical analyses. Immunohistochemical examinations were performed after fixation with 4% paraformaldehyde and staining with anti-CD31 antibody (Pharmingen, BD Biosciences, Le Pont de Claix, France), using an AxioImager M2 microscope (Carl Zeiss, Jena, Germany). Segmentation of tissues was performed using a fully connected, three-layer back-propagation neuronal network, as previously described [12] .
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