Protocol Citation: Jenny Eng, Koei Chin, Zhi Hu 2022. Cyclic Immunofluoresence Staining Protocol (OHSU). protocols.io https://dx.doi.org/10.17504/protocols.io.23vggn6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 22, 2019
Last Modified: February 18, 2022
Protocol Integer ID: 23381
Keywords: Cyclic, Immunofloresence
Abstract
Cyclic immunofluorescence protocol, including tissue processing and staining, image acquisition and fluorophore bleaching.
Materials
MATERIALS
10mM Citrate Buffer: 2.1 g citrate monohydrate dH2O up to 1000mL 5M NaOH until pH is 6 (approximately 3-4 mL) filteredCatalog #C-1909
pH9 Tris/EDTA bufferCatalog #S2367
SlowFade™ Gold Antifade Mountant with DAPICatalog #S36938
AF555 NHS Ester MW=1250Catalog #A37571
AF750 NHS Ester MW=1300Catalog #A37575
30% H202 (Make 3% with 1 ml H202 40 ul 5M NaOH 1 ul 10x PBS and 7.96 ml filtered dH20)Catalog #H1009
Protocol materials
30% H202 (Make 3% with 1 ml H202 40 ul 5M NaOH 1 ul 10x PBS and 7.96 ml filtered dH20)Catalog #H1009
10mM Citrate Buffer: 2.1 g citrate monohydrate dH2O up to 1000mL 5M NaOH until pH is 6 (approximately 3-4 mL) filteredCatalog #C-1909
pH9 Tris/EDTA bufferCatalog #S2367
SlowFade™ Gold Antifade Mountant with DAPICatalog #S36938
AF555 NHS Ester MW=1250Catalog #A37571
AF750 NHS Ester MW=1300Catalog #A37575
Target Retrieval Solution, pH 9 (10X)Catalog #S2367
10mM Citrate Buffer: 2.1 g citrate monohydrate dH2O up to 1000mL 5M NaOH until pH is 6 (approximately 3-4 mL) filteredCatalog #C-1909
30% H202 Catalog #H1009
Tissue Preparation
Tissue Preparation
For phospho-protein preservation, fix tissues in formalin at 4 degrees C for 12-24 hours. Use a standard histopathological protocol for clearing, dehydration and paraffin processing and embedding. Cut 4-5 um sections of formalin-fixed parrafin-embedded tissues and place on Tanner Adhesive Slides (Mercedes Medical, TNR WHT45AD)
Koei: For phospho-protein preservation, fix tissues in formalin for 12-24 hours immediately after resection. Keep tissues at 4 degrees C from resection through fixation...
2d
Bake slides 12-16 hours at 55 degrees C plus 30 minutes at 65 degrees C
16h 30m
Deparafinization
Deparafinization
Deparaffinize and hydrate sections through xylenes and graded alcohols as follows, using the Sakura Tissue Tek II Manual Slide Staining Set or similar solvent-resistant containers.
43m
Xylenes 3 x 5 min
(Change xylene and 100% EtOH when visibly dirty; 1-4x per month, depending on usage)
15m
100% EtOH 2 x 5 min
(Change xylene and 100% EtOH when visibly dirty; 1-4x per month, depending on usage)
10m
95% EtOH* 2 x 2 min
*use fresh every time to ensure correct concentration
4m
70% EtOH* 2 x 2 min
*use fresh every time to ensure correct concentration
4m
dH2O water* 2 x 5 min
*use fresh every time to ensure correct concentration
10m
Antigen Retreival
Antigen Retreival
Perform antigen retreival in a medical (histopathology lab) grade pressure cooker. This protocol is specific to the Biocare Medical Decloaking Chamber Pro or Dako Pascal (Discontinued)
Settings: SP1: 125°C, 30 seconds
SP2: 90°C, 30 seconds
SP limit: 10°C
Target Retrieval Solution, pH 9 (10X)Catalog #S2367
10mM Citrate Buffer: 2.1 g citrate monohydrate dH2O up to 1000mL 5M NaOH until pH is 6 (approximately 3-4 mL) filteredCatalog #C-1909
1h
Fill chamber with 500 ml dH2O
Fill 1 plastic Coplin jar with 1x Target Retreval Solution (pH9 Tris/EDTA buffer: left over from my olkd protocol from UCSF), prepared with dH2O
Fill 1 plastic Coplin Jar with 10 mM Citrate buffer, pH6
Fill 1 Tissue Tek container with dH2O
Place slides in container with pH 6 buffer and fill in any blank spots with dummy slides to ensure even heating. Place all filled staining containers into pressure cooker chamber. Record the time.
Close the chamber and hit "start/stop" to begin SP1 -- temperature will rise to 125 degrees and hold for 30 seconds. When chamber beeps, record time and pressure for quality control. (Pressure will be about 15 psi)
15m
Hit "start/stop" to begin SP2 -- temperature will lower to 90 degrees and hold for 30 seconds. When the chamber beeps again, record the time and pressure (the pressure should be 0 psi).
25m
Release pressure by pushing on the knob on the lid and turn off instument.
Remove the lid and, going quickly to retain heat, use forceps to remove the slides one at a time from pH6 buffer, dip them once brielfy in the hot water in the Tissue Tek container, and place them in the pH9 buffer. Put the lid back on the chamber and leave the slides for 15 minutes.
15m
Rinse slides in 2 changes of dH2O to cool to room temperature. Post antigen retreval, do not allow the slides to dry at any time.
30s
Wash slides 1 x 5 min in PBS.
5m
Quenching
Quenching
Quench in 3% H2O2 for 30 minutes (also reduces tissue autofluorescence)
30% H202 Catalog #H1009
30m
Prepare 10 ml quenching solution per slide (make fresh each time?).
i. 7.96 mL MilliQ H2O
ii. 1 mL 10x PBS
iii. 40 μl 5M NaOH
iv. 1 mL 30% H2O2 - add last, right before using
Koei: 30% Store condition?
Add 10 mL quenching solution to each compartment of the plastic chamber (Item # ?).
Take slides out of PBS, tap off excessl, and place face down in quenching solution.
Turn on lamp and position right above slides, with chamber lid off.
Quench for 30 minutes to fully quench Cy2/AF488 signal.
30m
After 30 minutes, remove slides with forceps and rinse 3 x 2 min in 1x PBS in a Coplin jar.
6m
Blocking
Blocking
Block for 30 minutes with 10% NGS and 1% BSA in 1x PBS. For smaller tissues, cover with 50 ul of blocking buffer (no coverslip required if buffer completely covers tissue). For larger tissues, use 75-100 ul of blocking buffer and use a plastic coverlsip to cover and evenly spread the buffer over the tissue.
Koei: Ragents and materials
- Bovine Serum albumin (Sigma, A7906).
- Phosphate buffered saline, 10x solution (Fisher, BP399).
- Normal goat, rabbit serum (Vector lab, S-1000).
- Plastic cover film (Grace Bio-Lab).
30m
Prepare blocking solution: 10% NGS and 1% BSA in 1x PBS and apply to tissues. Cover with plastic coverslip is necessary ( IHC World, IW-2601).
5m
Incubate tisssues at room temperature in a humidity chamber for 30 minutes.
Staining
Staining
Stain tissue with direct-labelled primary antibodies conjugated to Alexa-Fluor dyes (AF488, AF555, AF647 and AF750)
Prepare primary antibodies diluted in 5% NGS, 1% BSA in 1x PBS. Use 25-100 μl depending on size of tissue
Apply primary antibody, cover with plastic coverslip, and incubate for 2 hours at room temp in humidity chamber, protected from light. (OR incubate overnight at 4°C for stronger staining)
16m
Soak briefly in PBS to removeplastic coverslip. Then, transfer slide to new Coplin jar filled with PBS.
Wash 3 x 5 min in PBS, protecting from light.
15m
Mount in Slowfade Gold DAPI mounting media (Life Technologies, S36938). Use compressed air to blow dust off coverslip before mounting. Use ~15μl mounting media for small coverslips and ~30μl for large coverslips. Carefully drain/blot off excess mounting media.
Koei:
- Cover glass #1.5 thickness, 24x30 mm and 24x50 mm (Corning Life Sciences, 2980-243 and 2980-245)
- Antifade Mountant with DAPI (Thermo, S36938)
Repeat
Repeat
1h 28m
1h 28m
After mounting, image section on microscope/scanner. Then, repeat Quenching (step 5) and Staining (Step 7), followed by imaging.
Perform full slide fluorescent scanning with the Zeiss AxioScan.Z1 or similar instument.
After a successful scan is obtained, remove coverslips by immersing slides in PBS for 5-30 minutes. Allow coverslip to slide off naturally, without manually pushing or pulling it.
30m
Transfer to a new Coplin jar filled with PBS. Wash 3 x 2 minutes in PBS to remove residual mounting media.
6m
Perform 30 minutes of quenching, as in step 5.
36m
Apply primary antibodies as in step 7.
16m
Repeat for desired number of rounds.