Kubke Lab:Research/CND/Records2010-2011Summer/MH002

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Cranial Nerve Development Experiment

Contents

Embryo details


Species: Gallus gallus domesticus
Embryo Name: MH002
Embryo stage: ST28 (confirmed by Fabiana)
Staging description:
Fixation: PF
Cryoprotection: Yes
Material label and storage: Stored in a vial containing PBS in 30% Sucrose solution overnight at room temperature ON THE 2/12. On the 3/12 the embryo was stored in the histology lab freezer. The slides are labelled MH002 and stored in a brown folder in Fabiana's lab.

(Note: can you please provide information as to where the slides are stored/labelled?--MF Kubke 04:28, 3 February 2011 (EST))- Done

Experiment details


Objective: To determine what are the best parameters to cut a stage 28 embryo.
Procedure:

(Note: can you please confirm that the knife angle was 0? I thought we didnt start using that knife angle until later--MF Kubke 04:27, 3 February 2011 (EST))(Satya had left it on 0 as a "default" knife angle. It was moved just above 0 by you (approximately 0.5). I will check my notes to be sure .)--Malisha Hettiarachchi 16:02, 3 February 2011 (EST)
(Note: Can you please confirm this? It was my understanding you guys were cutting with a knife angle of about 15 degrees. I remember lowering the angle mid December - Please consult with Satya what her default angle is --MF Kubke 17:04, 3 February 2011 (EST))

Cryostat Sectioning Cryostat settings (for a more detailed protocol visit Kubke_Lab:Cryomicrotomy)

Cryostat Histology Lab
Knife15
Day Cut3/12/2010
Knife Angle
Chamber Temp-23C
Object Temp-23c
Glass SlidesPolysine slides
Plane of sectionCoronal
Number of slides8
ObservationsDue to the humidity the sections would immediately begin curling when on the metal platform. A razor blade was used to try catch the section therefore not allowing it to curl. This was later disused as a technique as it may have caused additional tissue damage.

(Include in your observations, eg ,were the sections serial, was any section lost, was quality assessed, etc)




Comments: Incubated the mould in the cryostat for 30 minutes at a temperature of -23 C OT/CT before placing it on the chuck

(Note: what did you incubate? I dont understand this entry--MF Kubke 04:35, 3 February 2011 (EST))

Results

  • H&E stained sections following this procedure.
  • Fabiana's feedback: Large holes in the sections which are not expected. Cannot determine if the issue is with staining or sectioning. ( Corresponding to the following entry) --Malisha Hettiarachchi 19:27, 31 January 2011 (EST).
(Note: You need to provide the staining details --MF Kubke 04:29, 3 February 2011 (EST))

Images

Summary

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