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, Les larves prélevées sont fixées en solution de Bouin dans un pilulier

, Après 48H d'exposition, rincer abondamment les larves avec de l'alcool à 70° jusqu'à ce que la solution reste claire

, Conserver les larves en alcool 70° dans un contenant hermétique pour stockage

, La paraffine étant hydrophobe, il faut déshydrater avec des bains successifs d'alcool de degrés croissants, puis remplacer l'alcool par un solvant miscible avec la paraffine

, Les larves se conservent indéfiniment dans l'alcool 70° dans un récipient hermétique à l'abri de la lumière et des variations de température

. Une,

, Positionner le bloc de manière à ce que la face du trapèze soit parallèle à la lame. 2. Régler l'angle du microtome à 5°, mettre le mode TRIM réglé à une épaisseur de 10µm

, Lancer la découpe et attendre l'apparition de la larve pour passer en mode SECT/CONT, réglé à une découpe de 7µm et une vitesse comprise entre 3 et 5 (selon préférences)

, Lors de la découpe, la paraffine doit fournir un ruban peu strié et non percé

, Le ruban obtenu doit être placé sur un papier noir à dessin

, Avant la phase de collage, s'assurer d'avoir dégraissé les lames avec une solution d'alcool à 95° et 1% d'acide acétique avant de les avoirs rincés à l'eau distillée et mise à sécher à l'étuve

, Couper le ruban à l'aide d'un rasoir, en morceaux d'une longueur équivalente à environ la moitié de celle de la lame

, Eliminer les parties ne possédants pas de coupes de la larve

, Poser une lame, préalablement annotée, sur la plaque chauffante (60°C) et y déposer une couche de solution d'albumine à 1% sur toute la surface

, Déposer très délicatement le morceau de ruban au milieu de la lame recouverte de la solution. Le ruban va ainsi s'étirer

, Attendre quelques secondes l'étirement total du ruban avant de récupérer la lame

, Poser la lame sur une feuille de papier absorbant et faire en sorte d'éliminer l'albumine par capillarité sans déplacer le morceau de ruban

, A l'aide d'un morceau de papier absorbant

, Les colorants étant en solution aqueuse, il faut éliminer la paraffine et réhydrater les lames pour pouvoir les colorer. Après coloration, on déshydrate par des bains d'alcool et de xylène avant le montage

A. Déparaffinage, Suite au séchage en étuve, récupérer les lames et mettre la plaque chauffante à chauffer à une température supérieure au point de fusion de la paraffine (60°C)

, Poser la lame sur la plaque environ 5 secondes le temps à la paraffine de fondre

, Xylène Après l'étuve, les lames peuvent être conservées à l'abri de la poussière et de la lumière, Placer les lames dans le porte-lames avant de réaliser les différents bains de déparaffinage 3

, Réaliser trois bains de 5 minutes

, Alcool 100° Réaliser trois bains de 5 minutes

, Alcool 95° Réaliser trois bains de 5 minutes

, Alcool 70° Réaliser trois bains de 5 minutes

, Eau distillée Réaliser trois bains de 5 minutes

P. Préparer-deux-tubes-eppendorf, U. Noté-«--d-», and . +d-»,

, Ajouter 190µl de solution de réaction ADN (voir partie « Solutions ») dans chaque tube

, Ajouter 10µl de solution standard (fournie dans le kit) de faible concentration en ADN (0 ng/µg) dans le tube

. L'extrait-«-analyse,

, Protocole d'analyse ARN/ADN

J. Denis and J. Di,

, Ajouter 10µl de solution standard (fournie dans le kit) de forte concentration en ADN (10 ng/µg) dans le tube « +D

, Préparer deux tubes Eppendorf PCR, un noté « -R » et l'autre « +R

, Ajouter 190µl de solution de réaction ARN (voir partie « Solutions ») dans chaque tube

, Ajouter 10µl de solution standard (fournie dans le kit) de faible concentration en ARN (0 ng/µg) dans le tube « -R »

, Ajouter 10µl de solution standard (fournie dans le kit) de forte concentration en ARN (10 ng/µg) dans le tube « +R

L. Quantité-d'extrait, celle de solution de réaction à introduire dans le tube « Analyse » est dépendante de la morphologie de l'espèce. En effet, pour une larve de grande taille, la quantité d'ADN et d'ARN contenue dans une certaine quantité d'extrait sera plus élevée que pour une larve de petite taille

, Transférer 1-20 µl d'extrait du tube « Analyse » dans chaque tube PCR ADN correspondant

, Ajouter 180-199µl de solution de réaction ADN

, Transférer 1-20 µl d'extrait du tube « Analyse » dans chaque tube PCR ARN correspondant

, Ajouter 180-199µl de solution de réaction ARN

, Mesurer l'ADN et L'ARN au fluorimètre Qubit et renseigner la feuille pour chaque larve

, Une fois les valeurs de concentrations obtenues, soustraire la quantité (en ng/ml) d'ARN par celle d'ADN

, Au cas cela peut être nécessaire, le reste de surnageant des tubes « Analyse