Immunofluorescence Procedure Tu 9/26/23

Materials

• PBS-Tw-Az for rinsing
• Little beakers
• forceps
• enough for 4 humid chambers
  • petri dish
  • filter paper
  • parafilm
• fixed coverslip of HTC116 cells, 1 per student
• Mixed primary antibodies
  • Mouse anti-Lamin A
  • Rat anti-alpha tubulin
• Mixed secondary antibodies
  • Goat anti mouse 488 (green)
  • Goat anti rat TRITC (red)

To mix primaries:

• Lamin A:
  • Use 4 aliquots of 5 µL each
  • Add 125 µL PBS-Tw-Az-BSA to get (slightly less than) 2X strength
  • total volume: 520 µL
• Alpha Tubulin:
  • Use 3 aliquots of 4 µL each
  • Add 200 µL PBS-Tw-Az-BSA to get 2X strength
  • total volume = 600 µL
• Mix 1:1, and aliquot ~104 µL per pair. Make 10 aliquots

To mix secondaries

• Green anti-mouse:
  • 2 1µL aliquot
  • 300 µL PBS-Tw-Az-BSA
  • total volume: 602 µL
• Red anti-rat:
  • 2 5 µL aliquots
  • 260 µL PBS-Tw-Az-BSA each
  • total volume: 530 µL
• Mix 1:1, and aliquot ~106 µL per pair. Make 10 aliquots

Live Cell Staining & Imaging

Students use the dish they seeded previous Thursday.

Need "staining solution":

• Fluorobrite
• Nuc Blue
• Mitotracker Red

To make staining solution for	coverslip bottom dishes
with	mL staining solution per dish
Start with:	mL Fluorobrite
add:	µL Nuc Blue
and	µL Mitotracker Red

Kate transfects HCT116 cells with Eb1 plasmids.

Need 8-10 viewing dishes, seeded with transfected cells 1 to 2 days before viewing.

1 reaction needs ~ 10⁶ cells/mL

100 uL cells per reaction (barely 8 drops)

So do 2 reactions, each with 10⁶ cells.

2 days before, seed a 25 cm² flask with 0.5 10⁶cells.

(4 days before, seed with 0.125 10⁶ cells)

1 drop of the 100 uL reaction into its own dish.

On transfection day, divide into 2 reactions

Notes on actual protocol followed:

Friday 10/6

• Start with 2 confluent 12.5 cm² flasks
  • (~1.25 x 10⁶ cells)
  • Need 1-5 x 10⁶ cells/mL on Monday
  • Split 1/8 to allow for 3 doublings
  • Stop reaction with 1.6 mL
  • Seed 0.2 mL into each of 2 new flasks.

Monday 10/9/23

• Put all the cells from one confluent 12.5 cm² flask
  • should be confluent again
• into a new 25 cm² flask

Tuesday 10/10/23

• There should be 2.5 x 10⁶ cells
• Trypsinize
• Stop reaction with 2 mL
• split suspended cells into two microfuge tubes.
• Spin down, and proceed with 2 Mirus nucleofection reactions
• Make 2 12.5 cm² flasks

Trial run for Lamin antibodies

Fix 15 min in paraglut

1^o Ab: Rabbit anti-lamin B

• dilute 1:1000 with PBS-Tw-Az-BSA
• 2 µL antibody into 2 mL buffer
• 1 hr 37C

2^o Ab: goat anti-rabbit

• 1 hr RT incubation

Cell type	# coverslips
MEF WT NLS-GFP (40)	3
HCT-116 WT (60)	3
DU-145 (74)	4

Stain with Hoechst (1:10,000) in the final PBS-Tw-Az rinse

Mount with non-DAPI Fluoromount (Fisher OB100-01)

Students begin projects

Kate provides fixed coverslips (seeded 10/22, fixed 10/24/23)

Cell line	medium	variant	treatment	no. coverslips
HCT116	McCoy	WT (60)		4
		LB1-AID-GFP (68)		2
MEF	DMEM	WT (39)	control	6
			VPA
			DZNP
		LMNB1-/- (02)		3
	DMEM+G418	LA KD (37)		3
Prostate cancer	RPMI-1640	DU145 (74)		2 +3 from Mai
	RPMI-1640	LNCaP (77)		2 + 3 from Mai
	DMEM	PC3 (76)		2

Materials:

• Paraglut fix (enough for 30 wells)
• PBS to rinse 30 coverslips
• PB-TW-Azide (enough for 24 dunking beakers and 30 wells)
• 1^o antibodies
  • mouse anti-NP (nuclear pore)
  • mouse anti-LB1
  • rabbit anti-LB2 ab151735 (get one 10 µL aliquot from Drew - dilute 1/1000)
• 2^o antibodies
  • goat anti-mouse green for NP
  • goat anti-rabbit red for LB1 & LB2
• Slides
• slide labels
• DAPI & droppers
• forceps
• dunking beakers
• humid chamber materials

Project	people	scope	IF coverslips	IF details
MEF VPA	Olivia & Juliana	1	1x 39 unt, 1X 39 VPA, another VPA if pos	standard NP LB1 IF
MEF DZNep	Karan & Nate	2	1x 39 unt, 1X 39 DZNep, another if pos	standard NP LB1 IF
MEF LMNB1-/-	Cole & Grif	3	1X 39, 2X 02 LMNB1-/-	39 & 02 NP LB2 IF, 1x 02 NP LB1 IF
MEF LA KD	Shrushti, Emma, Anna	8	1X 39, 2X 39	standard NP LB1 IF
HCT116 LB1 live 1	Sam & Jillian	5	68, 1x unt & 1x VPA	LB2 IF only
HCT116 LB1 live 2	Isabelle & Jason	6	60, 1x unt 1x VPA; 60, 1x unt 1x VPA	one set NP LB1 IF; other set NP LB2 IF
Prostate Cancer 1	Tyian & Antonia	4	2x 74, 1x 76	standard NP LB1 IF
Prostate Cancer 2	Kelsey & Pedro	7	2x 74, 1x 77	standard NP LB1 IF