2024 Bioimaging (Stephens)

2024 Bioimaging (Stephens) kdorfman Mon, 08/12/2024 - 22:37

HeLas for teaching microscopy

MEFs for CURE

day date prep
Tu 9/3 12 coverslips: HeLa (microtubules, actin, DAPI)
Th 9/5 (Transmitted light and alignment)
Tu 9/10 (Cameras, SNR, calibration)
Th 9/12 (NA and resolution) beads
Tu 9/17 (Fluorescence)
Th 9/19 photobleaching (need new slides?)
Tu 9/24 IF: need fixed coverslips, 10, 20 Ab
Th 9/26 view IF from previous class, cell culture (MEF 39)
Tu 10/1 Live cell imaging and cell culture
Th 10/3 Plasmid prep Eb1 into MEF 39 and cell culture
Kate transfects, puts into live dishes
Tu 10/8 Transfection imaging
Th 10/10 midterm
Tu 10/15 NO CLASS (Monday schedule)
Th 10/17 Begin CURE
Tu 10/22 need 6 HeLa coverslips for IF
rabbit Lamin B1, green
mouse Emerin red
Th 10/24
Tu 10/29
Th 10/31
Tu 11/5 NO CLASS (Election Day)
Th 11/7
Tu 11/12
Th 11/14
Tu 11/19
Th 11/21
Tu 11/26 CURE written report due
Th 12/3 (Michael) Neurobiology
Tu 12/5 (Michael) Neurobiology
Tu 12/10 Flexible last day of class
Bioimaging 477H Syllabus Fall 2024 v240829.pdf

IF for 9/24

IF for 9/24 kdorfman Wed, 08/28/2024 - 20:47

Fixed coverslips (MEF 39 from Drew's lab)

Fix in paraformaldehyde

primary secondary
Lamin B1 (rabbit, 1:1,000) green
NPC (mouse, 1:2,000) red
Emerin (mouse, 1:1,000) (from Drew's lab) red

To make the double primaries:

Make 2x Lamin B1 (add 1 mL instead of 2 mL)

Make 2x NPC (follow label directions = 2x (should say 1:20)

Make 2x Emerin: 1:500

Mix 250 µL each

To make the double secondary:

  • Make 2x goat anti rabbit green
    • goat anti rabbit green aliquots need 1194 µL to be 1X
    • make 2X by adding 597 µL PBS-Tw-Az BSA
  • Make 2X goat anti mouse red:
    • aliquot says add 500 µL to make 1X
    • add 250 µL to make 2X (use 2 aliquots)
  • Make 1x green + red by mixing
    • 500 µL goat anti rabbit green with
    • 500 µL goat anti mouse red

DAPI

Slides

Beakers

forceps

Humid chambers

PBS-Tw-Az