Slides kdorfman Tue, 12/27/2011 - 20:56

slides 2012

slides 2012 kdorfman Fri, 08/23/2013 - 14:07



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


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


12 for Lab 3.3, unmounted

slides 2013

slides 2013 kdorfman Fri, 08/23/2013 - 14:08
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

slides 2015 kdorfman Tue, 07/14/2015 - 14:58

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

organelle slides kdorfman Tue, 07/14/2015 - 14:59

Alpha actinin

Alpha actinin kdorfman Tue, 07/14/2015 - 15:02

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

Gamma tubulin kdorfman Tue, 07/14/2015 - 15:03

Stains Centrosomes

  • Monoclonal, GTU-88, Sigma T6557
  • Fixation: methanol; para/glut
  • Dilution 1:100

From Sigma:

General description

γ-Tubulin (48kDa) is a ubiquitous and highly conserved protein within the microtubule organizing centers (MTOCs) in eukaryotic cells. γ-Tubulin is mapped to human chromosome 17q21.2 and codes for a member of the tubulin family. Human TUBG1 transcript is widely expressed in preimplantation embryos and brain. Monoclonal Anti-γ-Tubulin (mouse IgG1 isotype) is derived from the GTU-88 hybridoma produced by the fusion of mouse myeloma cells and splenocytes from an immunized mouse.


Monoclonal Anti-γ-Tubulin recognizes an epitope located in the N-terminal amino acids of γ-tubulin (48 kDa). Cross reactivity has been observed with human, bovine, dog, hamster, rat, mouse, chicken, and Xenopus γ-tubulin. The antibody recognizes an epitope located within the N-terminal region of γ-tubulin.


synthetic γ-tubulin peptide, conjugated to KLH


Monoclonal Anti-γ-Tubulin antibody has been used in western blotting,[1] indirect immunofluorescence (IF) and immunofluorescence staining. Monoclonal Anti-γ-Tubulin is also suitable for use in immunochemical applications such as immunoblotting, immunocytochemical staining of cultured cells and in ELISA. Monoclonal Anti-gamma-Tubulin is suitable for use in immunochemical applications such as immunoblotting, immunocytochemical staining of cultured cells, and in ELISA.

Biochem/physiol Actions

γ-Tubulin nucleates microtubule assembly throughout the mammalian cell cycle in vivo. γ-Tubulin binds microtubule minus ends and is responsible for mediating the link between microtubules and the centrosome. It functions as the microtubule nucleator at the MTOC. It binds to the β-tubulin half of the tubulin molecule, thus establishing the polarity of a microtubule, leaving the α-tubulin half exposed at the plus end. γ-Tubulin abundance is less than 1% of the level of either α- or β-tubulin. Overexpression of γ-tubulin is observed in lung cancer. The expression levels of γ-tubulin can be considered as an important prognostic indicator for patients with astrocytomas.

Physical form

The product is provided as ascites fluid with 15 mM sodium azide as a preservative.

Storage and Stability

For continuous use, store at 2-8 °C for up to one month. For extended storage, freeze in working aliquots. Repeated freezing and thawing, or storage in "frostfree" freezers, is not recommended. If slight turbidity occurs upon prolonged storage, clarify the solution by centrifugation before use.


Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Golgi 58K

Golgi 58K kdorfman Tue, 07/14/2015 - 15:04
  • Sigma G2404, mouse monoclonal
  • Formaldehyde (no glut)
  • 1:100


Hec1 kdorfman Tue, 07/14/2015 - 15:05

Stains kinetochores

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


LAP2 kdorfman Tue, 07/14/2015 - 15:07

Stains nuclear envelope

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


Vinculin kdorfman Tue, 07/14/2015 - 15:08

Focal adhesion plaques

  • Monoclonal, sigma V4505
  • Formaldehyde
  • 1:100