Immunostaining of Cochlear Samples

For cochlear samples, P3 temporal bones (n = 1 for the WT, n = 2 for the mutant) were dissected and fixed at room temperature for 2 h or at 4°C overnight and then decalcified overnight at 4°C in a solution of 10% EDTA/PBS. The samples were then embedded in sucrose/gelatin, as described elsewhere (Mansour et al., 2013 (link)). Sections (thickness: 14 µm) prepared in the modiolar plane were stained with the following primary antibodies: rat anti-CD44 (550538; 1:1,000; BD Biosciences), rabbit anti-MYO7A (25-6790; 1:1,000; Proteus Biosciences), rabbit anti-p75NTR (#07-476; 1:650; Millipore), or goat anti-PROX1 (AF2727; 1:200; R&D Systems). The secondary antibodies were Alexa Fluor 594 goat anti-rat (A11007; 1:400; Invitrogen), Alexa Fluor 488 goat anti-rabbit (A11034; 1:400; Invitrogen), or Alexa Fluor 488 donkey anti-goat (A11055; 1:400; Invitrogen). DAPI was included in the mounting medium (Vectashield; Vector Labs).

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Cornille M., Moriceau S., Khonsari R.H., Heuzé Y., Loisay L., Boitez V., Morice A., Arnaud E., Collet C., Bensidhoum M., Kaci N., Boddaert N., Paternoster G., Rauschendorfer T., Werner S., Mansour S.L., Di Rocco F., Oury F, & Legeai-Mallet L. (2022). FGFR3 overactivation in the brain is responsible for memory impairments in Crouzon syndrome mouse model. The Journal of Experimental Medicine, 219(4), e20201879.

Publication 2022

rat anti-CD44 rabbit anti-p75NTR goat anti-PROX1 Alexa Fluor 594 goat anti-rat Alexa Fluor 488 goat anti-rabbit A11034 Alexa Fluor 488 donkey anti-goat A11055 Vectashield

Alexa 594 Alexa fluor 488 Antibodies Cd44 Cochlear Dapi Donkey Edta Gelatin Goat P75ntr Proteus Rabbit Sucrose Temporal bones Vector

Corresponding Organization :

Other organizations : Université Paris Cité, Inserm, Institut des Maladies Génétiques Imagine, Institut Necker Enfants Malades, Sorbonne Paris Cité, Hôpital Necker-Enfants Malades, Assistance Publique – Hôpitaux de Paris, De la Préhistoire à l'Actuel : Culture, Environnement et Anthropologie, Université de Bordeaux, Ministère de la Culture, Hôpital Lariboisière, Centre National de la Recherche Scientifique, ETH Zurich, Board of the Swiss Federal Institutes of Technology, University of Utah, Hospices Civils de Lyon, Hôpital Femme Mère Enfant, Université Claude Bernard Lyon 1

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Variable analysis

independent variables

Genotype (wild-type vs. mutant)

dependent variables

Protein expression levels of CD44, MYO7A, p75NTR, and PROX1 in cochlear samples

control variables

Age of temporal bones (P3)
Fixation time (2 h at room temperature or overnight at 4°C)
Decalcification time (overnight at 4°C in 10% EDTA/PBS)
Embedding (in sucrose/gelatin)
Section thickness (14 µm)
Staining with primary and secondary antibodies

controls

Positive control: not explicitly mentioned
Negative control: not explicitly mentioned

Annotations

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