Slides

slides 2012

CHECK THIS

LLCPk-1

coverslips, fixed in 3.5% formaldehyde and stored in PBS-tween-azide in coplin jars in the refrigerator:

  • 30 unmounted,

  • 10 mounted, stained only with DAPI

  • 20 stained for tubulin, and mounted with DAPI

  • 20 stained for actin and tubulin, and mounted with DAPI

  • 10 serum starved, stained for tubulin, mounted with DAPI

    • for Lab 4.1
    • Grow first in regular medium (otherwise they won't stick)
    • Replace medium with serum free medium.
    • Fix after 24 hours
  • 10 Arrested with 100nM nocodazole (8 - 16 hours)

    • for Lab 4.1
    • Treat with nocodazole late in the day
    • Fix the next day in para-glut or formaldehyde
    • Stain for tubulin, mount with DAPI

Nocodazole stock is 33 mM in DMSO
Make 100 µL 1 mM: 3 µL (33mM) + 97 µL medium
Make 1 mL 6µM: 6 µL (1mM) + 994 µL medium
Add 1 drop (~50 µL) 6µM to each dish of ~3 mL

OR

Make 30 mL 100 nM: 3 µL (1mM) in 30 mL medium
Change the medium in each dish.

3t3

12 for Lab 3.3, unmounted

slides 2013

Lab date # DAPI + fix
1.1 & 1.3 9/4 & 11 10 paraglut
1.2 9/9 10 alpha tub paraglut
2.1 & 2.2 9/16 10 alpha tub, actin paraglut
3.1 9/23 10 alpha tub, actin paraglut
3.2 9/25 2 Golgi formaldehyde (no glut)
2 gamma tubulin paraglut or methanol
2 actin paraglut
2 alpha actinin formaldehyde (no glut)
2 ZO1 methanol or paraformaldehyde
2 LAP2 methanol
2 vinculin formaldehyde

slides 2015

LL's for student slides

Fix 90 in formaldehyde; 10 in methanol

Plate 102 in 17 6-well plates.

Lab # coverslips fix stain secondary
1.1 10 form or paraglut
1.2 10 "
1.3 10 "
2.1 10 " anti-tubulin, phalloidin goat anti-rat
2.2 10 " anti-tubulin goat anti-rat
3.1 10 " anti-tubulin, phalloidin FRESH goat anti-rat
3.2 3 form alpha actinin anti-mouse IgM
3.2 3 form anti Golgi anti-mouse IgG
3.2 3 form vinculin
3.2 3 MeOH anti gamma-tubulin
3.2 3 " anti-Hec1
3.2 3 " LAP2
3.3 20 form or paraglut un-mounted for students to stain

organelle slides

Alpha actinin

Stains adhesions and stress fibers

  • Monoclonal, BM 75.2
  • Fix in Formaldehyde, no glut or meOH
  • 1:200
  • IgM, use mouse FITC secondary that recognizes IgMs

Gamma tubulin

Stains Centrosomes

  • Monoclonal, GTU-88, Sigma,
  • Fixation: methanol; para/glut
  • Dilution 1:100

Golgi 58K

  • Sigma G2404, mouse monoclonal
  • Formaldehyde (no glut)
  • 1:100

Hec1

Stains kinetochores

  • Novus biological, recombinant human HEc1, monoclonal
  • Fix in methanol
  • 1:200

LAP2

Stains nuclear envelope

  • Methanol
  • 1:100
  • BD transduction
  • Mouse IgG

Vinculin

Focal adhesion plaques

  • Monoclonal, sigma V4505
  • Formaldehyde
  • 1:100