Immunostaining - Revision history

Admin: /* PEM Buffer: */

2024-09-17T11:37:24Z

PEM Buffer:

← Older revision		Revision as of 11:37, 17 September 2024
Line 27:		Line 27:
	===== PEM Buffer: =====		===== PEM Buffer: =====

	* 80 mM PIPES pH 6.8		* 80 mM PIPES pH 6.8 (2.419g/100 mL)
	* 5 mM EGTA		* 5 mM EGTA (190 mg/100 mL)
	* 2 mM MgCl<sub>2</sub>		* 2 mM MgCl<sub>2</sub> (19 mg/100 mL)

	===== Antibody Buffer: =====		===== Antibody Buffer: =====

Admin at 13:04, 2 March 2022

2022-03-02T13:04:10Z

← Older revision		Revision as of 13:04, 2 March 2022
Line 5:		Line 5:
	== Protocol ==		== Protocol ==

	#Fix cells in 4% PFA/PBS for 20' at 4~~-10C~~.		#Fix cells in 4% PFA/PBS for 20' at room temperature. [https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC4958280/ To preserve membrane structures], fix with 4% PFA in PEM, 37C, 10 min.
	#Wash 3X PBS		#Wash 3X PBS
	#Permeabilize and block using antibody buffer, 1hr at RT.		#Permeabilize and block using antibody buffer, 1hr at RT.
Line 12:		Line 12:
	#**Put 100ul antibody mixture on a piece of parafilm		#**Put 100ul antibody mixture on a piece of parafilm
	#**Flip coverslip onto drop		#**Flip coverslip onto drop
	#**Cover with foil or box and leave undisturbed (KEEP LEVEL).		#**Cover with foil or box and leave undisturbed for 20 min (surface staining) or up to overnight (intracellular staining; minimum 1 hr). '''KEEP LEVEL'''.
	#Was 3X 15min with PBS.		#Was 3X 15min with PBS.
	#Add 1:~~200 secondary antibody~~ in antibody buffer:		#Add secondary antibody at desired concentration (typically 1:500 to 1:1000) in antibody buffer:
	#*Use hanging drop method:		#*Use hanging drop method:
	#**Cover with foil and leave undisturbed for 1-2 hours<br>		#**Cover with foil and leave undisturbed for 1-2 hours<br>
Line 24:		Line 24:

	== Recipes ==		== Recipes ==

			===== PEM Buffer: =====

			* 80 mM PIPES pH 6.8
			* 5 mM EGTA
			* 2 mM MgCl<sub>2</sub>

	===== Antibody Buffer: =====		===== Antibody Buffer: =====

Admin: Created page with "This procedure was optimized using the 4G10 (anti-phosphotyrosine) antibody, but should work well for many other antibodies. For a generic protocol, add an additional 20-60min..."

2021-02-01T19:56:21Z

Created page with "This procedure was optimized using the 4G10 (anti-phosphotyrosine) antibody, but should work well for many other antibodies. For a generic protocol, add an additional 20-60min..."

New page

This procedure was optimized using the 4G10 (anti-phosphotyrosine) antibody, but should work well for many other antibodies. For a generic protocol, add an additional 20-60min at RT fixing step after the 4C fixing step. New fixative is not needed, simply move the plate to the bench & cover with foil. Antibody concentrations will need to be optimized for each antibody.  Please enter details for all optimized antibodies in the table at the end of the protocol. 

 

== Protocol ==

#Fix cells in 4% PFA/PBS for 20' at 4-10C.
#Wash 3X PBS
#Permeabilize and block using antibody buffer, 1hr at RT.
#Add primary antibody at desired concentration in antibody buffer, 20C for 1 hour or 4C overnight.
#*Use hanging drop method:
#**Put 100ul antibody mixture on a piece of parafilm
#**Flip coverslip onto drop
#**Cover with foil or box and leave undisturbed (KEEP LEVEL).
#Was 3X 15min with PBS.
#Add 1:200 secondary antibody in antibody buffer:
#*Use hanging drop method:
#**Cover with foil and leave undisturbed for 1-2 hours 
#Wash 3X 15min in PBS.

Cell can be imaged in PBS, or mounted using DAKO on a slide. Imaging is generally better in PBS, as DAKO can distort cells as it dries.

 

== Recipes ==

===== Antibody Buffer: =====

PBS + 0.1% triton X-100 + 5% donkey serum or 2.5% BSA (use goat serum of secondary is derived in goats).

''For 10ml:''

*9.5ml PBS
*10ul Triton X-100 (exclude if surface-staining)
*500ul serum or 250mg BSA

 

Note: 1% BSA can be used in place of serum in many cases, especially after step 3.

 

== Working Conditions ==

{| width="100%" cellspacing="1" cellpadding="1" border="1"
|-
| '''Antibody '''
| '''Fixation''' 
| '''Primary Conditions '''
| '''Washes''' 
| '''Secondary Conditions''' 
| '''Washes '''
|-
| 4G10 
| 4% PFA, 20', 4C 
| 1:100 in antibody buffer, overnight, 4C 
| 3 x 15min PBS 
| 1:200 anti-mouse in antibody buffer, 1 to 2 hours, room temp. 
| 3 x 15min PBS 
|-
| 4G10 
| 4% PFA, 20', 4C 
| 1:100 in antibody buffer, 4hrs, 20C 
| 3 x 15min PBS 
| 1:200 anti-mouse in antibody buffer, 1 to 2 hours, room temp. 
| 3 x 15min PBS 
|-
| THE His6 Antibody ([http://www.genscript.com/antibody/A00186-THE_sup_TM_sup_His_Tag_Antibody_mAb_Mouse.html GenScript A00186]) 
| 4% PFA, 20', 4C 
| 1:250-1:500 in antibody buffer, 1-4hrs, 20C 
| 3 x 15min PBS 
| 1:1000 anti-mouse in antibody buffer, 1 to 2 hours, room temp. 
| 3 x 15min PBS 
|}