TITLE: MIBI and IHC solutions
AUTHORS: Marc Bosse, Christine Camacho, Sean Bendall, Mike Angelo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">These protocols are designed to prepare solutions to be used for the Multiplex Ion Beam Imaging Time of Flight instrument (MIBI_TOF) and IHC related protocols in the Bendall and Angelo labs. Particularly, the IHC protocol uses the same reagents and steps used for MIBI staining. Therefore, It can be used to validate the compatibility of a new target antibody before use for MIBI staining. These protocols have been successfully use for MIBI and are the result of extensive optimization experiments.  </div></div>

[STEPS]
?. [Target retrieval solution (Antigen retrieval)]
Make a fresh 1:10 dilution  of target retrieval solution 10x (3-in-1) pH 9 (Dako)in  of ultrapure (type 1,  >18 MOhms) water   ABC1Total volume (mL)Volume (mL) Target retrievalVolume (mL) H2O2252.522.535054541001090
2.5 mL
22.5 mL
ABC1Total volume (mL)Volume (mL) Target retrievalVolume (mL) H2O2252.522.535054541001090
?. [MIBI or IHC 1X PBS wash buffer]
Reuse the sam Nalgene 1L bottle if not expired.Note. The bottle is replaced monthly. New label name (14 pt font bold, format landscape)= MIBI 1x PBS wash buffer or IHC1x PBS wash buffer. Enter the expiring date 1 month from the present dateExp.: MM/DD/YYYY  AB1ReagentsQty for  1000 mL2PBS IHC Wash Buffer with Tween 20 (mL)503Bovine Albumin (BSA), heat shock treated (g)14Ultrapure (type 1) water (mL)949
AB1ReagentsQty for  1000 mL2PBS IHC Wash Buffer with Tween 20 (mL)503Bovine Albumin (BSA), heat shock treated (g)14Ultrapure (type 1) water (mL)949
?. [MIBI or IHC 1X PBS wash buffer]
Verify stock of MIBI or IHC 1x PBS wash buffer and prepare accordingly if running low
?. [MIBI or IHC 1X PBS wash buffer]
Use the same 1 L Nalgene Bottle, if before expiring date. Replace by a new 1 L Nalgene bottle on monthly basisUse single use 50 mL tube for measurementUse the measurement marks on the bottle to complete with waterDo not use any other vessels than single use graduated plastic containers to prevent cross contamination
?. [MIBI or IHC 1X PBS wash buffer]
?. [Antibody Diluent ]
Prepare Antibody Diluent (NHS 3%)
?. [MIBI or IHC 1X PBS wash buffer]
Prepare 1x PBS wash buffer as follow:Add reagent in row 2 and row 3 first, mix gently than complete with row 4, ultrapure (type 1, >18 MOhms) water  AB1ReagentsQty for  1000 mL2PBS IHC Wash Buffer with Tween 20 (mL)503Bovine Albumin (BSA), heat shock treated (g)14Ultrapure (type 1, >18 MOhms) water (mL)949
AB1ReagentsQty for  1000 mL2PBS IHC Wash Buffer with Tween 20 (mL)503Bovine Albumin (BSA), heat shock treated (g)14Ultrapure (type 1, >18 MOhms) water (mL)949
?. [Gelatin 10% stock, 100x]
Prepare stock 10% weight/ volume cold fish water gelatin
?. [Triton 10% stock, 100x]
Prepare Triton stock 10% volume/ volume
?. [MIBI or IHC 1X PBS wash buffer]
Label name (Brady label 0.5", M21-500-7425,  Font 14 pts bold, format: Banner landscape) First line: MIBI or IHC 1X PBS wash bufferSecond line: Exp.: MM/DD/YY (Expiring date, 1 month from the present date)
?. [Antibody Diluent ]
Add the stock solution  of TBS IHC Wash Buffer with Tween 20  (Cell Marque, 20x)
7.5 mL
?. [Antibody Diluent ]
Make some small aliquots by distributing 1 mL with a 10 mL serological pipette in 1.5 or 2 mL tubes and stock aliquots of 10 mL in 15 mL tubes
?. [Antibody Diluent ]
Make a 3% (volume/volume) of normal Horse serum Add Normal Horse Serum (Vector labs, S-2000) in  of Antibody diluent preparation buffer (1xTBS-Tween) in 50 mL tube
1.5 mL
48.5 mL
?. [Antibody Diluent ]
FIlter with a 50 mL syringe  (Fisher Scientific, 13-689-8) and filter (Millipore, Millex-HV, SLHV033RS) into a new 50 tube
?. [Antibody Diluent ]
Label cap tubes using dot printed labels Antibody Diluent (NHS 3%)
?. [Antibody Diluent ]
Store at -20˚C small aliquots in the staining box reagents and stock aliquots in the specified location
?. [MIBI or IHC 1X PBS wash buffer]
Store at 4˚CReuse the same Nalgene bottle to make new 1x PBS wash buffer until the initial expiring date (
?. [Antibody Diluent ]
Complete to  with UltraPure Water (Invitrogen, 10977-015)
150 mL
?. [Gelatin 10% stock, 100x]
Warm up the gelatin stock bottle at  prior use to reduce viscosity
37 °C
?. [Gelatin 10% stock, 100x]
Take gelatin by aspirating ~  using a large opening pipette tip >4 mm OD
1 mL
?. [Gelatin 10% stock, 100x]
Add slowly the viscous solution to the tube until it weigh
1 g
?. [Gelatin 10% stock, 100x]
Put a 15 mL tube on a precision balance and tare it to zero
?. [Gelatin 10% stock, 100x]
Take  of warm Ultrapure Water (Invitrogen, 10977-015) and add into the gelatin 15 mL
8 mL
?. [Gelatin 10% stock, 100x]
Make sure that all the gelatin is dissolved and complete the volume to
10 mL
?. [Gelatin 10% stock, 100x]
Make small aliquots by distributing ~1 mL with a 10 mL serological pipette in 1.5 or 2 mL tubes
?. [Gelatin 10% stock, 100x]
Store at -20˚C small aliquots in the staining box reagents
?. [Triton 10% stock, 100x]
Take Triton by aspirating slowly   using a large opening pipette tip >4 mm OD
1 mL
?. [Triton 10% stock, 100x]
Transfer into the  tube
50 mL
?. [Triton 10% stock, 100x]
Add  of  Ultrapure Water (Invitrogen, 10977-015) to a  tube
9 mL
50 mL
?. [Triton 10% stock, 100x]
Make sure that all the Triton is dissolved
?. [Triton 10% stock, 100x]
Make small aliquots by distributing ~1 mL with a 10 mL serological pipette in 1.5 or 2 mL tubes
?. [Triton 10% stock, 100x]
Store at -20˚C small aliquots in the staining box reagents
?. [Sodium azide (20 mg/mL), 100x]
Prepare 100x (2%) of sodium azide
?. [Sodium azide (20 mg/mL), 100x]
Weigh  of sodium azide
1 g
?. [Sodium azide (20 mg/mL), 100x]
Add of Ultrapure Water (Invitrogen, 10977-015) in a
48 mL
50 mL
?. [Sodium azide (20 mg/mL), 100x]
Add sodium azide in the  tube
50 mL
?. [Sodium azide (20 mg/mL), 100x]
Make sure that all the sodium azide is dissolved and complete the volume to
50 mL
?. [Sodium azide (20 mg/mL), 100x]
Make small aliquots by distributing ~1 mL with a 10 mL serological pipette in 1.5 or 2 mL tubes
?. [Sodium azide (20 mg/mL), 100x]
Store at 4˚C small aliquots in the reagents box
?. [Blocking Buffer]
Prepare the blocking buffer following the table:  ABCD1ReagentsQty for 10 mLQty for 30 mLQty for 50 mL21xTBS IHC Wash Buffer with Tween 20 (mL)9.528.547.53Triton 10% (mL)0.10.30.54Gelatin 10% (mL)0.10.30.55Horse serum (mL)0.20.616Sodium azide 20 mg/mL (mL)0.10.30.5
ABCD1ReagentsQty for 10 mLQty for 30 mLQty for 50 mL21xTBS IHC Wash Buffer with Tween 20 (mL)9.528.547.53Triton 10% (mL)0.10.30.54Gelatin 10% (mL)0.10.30.55Horse serum (mL)0.20.616Sodium azide 20 mg/mL (mL)0.10.30.5
?. [Blocking Buffer]
Filter the blocking buffer with a filter (Millipore, Millex-HV, SLHV033RS) into a new tube
?. [Blocking Buffer]
Make small aliquots by distributing ~1 mL with a 10 mL serological pipette in 1.5 or 2 mL tubes
?. [Blocking Buffer]
Store at -20˚C small aliquots in the staining box reagents
?. [Antibody Diluent ]
Prepare antibody diluent preparation buffer (1xTBS-Tween)Add  UltraPure Water (Invitrogen, 10977-015) in a Nalgene filter unit (Fisher Scientific, 09-740-28E)
100 mL
?. [Antibody Diluent ]
Filter under vacuum
?. [Antibody Diluent ]
Label as Antibody diluent preparation buffer (1xTBS-Tween)
?. [PBS 10x low barium]
Use a new 1 L Nalgene bottle
?. [PBS 10x low barium]
Add 900 mL of ultrapure (type 1, >18 MOhms) water in 1 L Nalgene bottle Use the measurement marks on the bottleDo not use any other vessel to measure the water
?. [PBS 10x low barium]
Weight the following chemicals:For 1 L add: NaCl KCl Na2HPO4.7H2OKH2PO4
80 g
2 g
26.8 g
2.4 g
Make sure that the chemical used correspond to the formula, particularly, for the sodium phosphate dibasic heptahydrate
?. [PBS 10x low barium]
Mix thoroughly. Do not adjust pH
?. [PBS 10x low barium]
Filter using 2 x 500 mL vacuum filter unit (Fisher Scientific, 09-740-28C)
?. [PBS 10x low barium]
Label each bottles with Brady lab printer (font 14, bold, banner landscape, tape 1/2") as:    First line:  PBS low barium 10x stock   Second line:  Date (MM/DD/YY)
?. [Tris Buffer pH 8.5 100mM]
Use a new 1 L Nalgene bottle, label with Brady lab printer (font 14pt, bold, banner landscape, 1/2" tape)  as follow:     First line: 100 mM TRIS BUFFER pH8.5    Second line: Exp.: MM/DD/YY  (expiration date: add 1 month to the preparation date in a new bottle)
?. [Tris Buffer pH 8.5 100mM]
Add 900 mL of ultrapure (type 1, >18 MOhms) water in 1 L Nalgene bottle Use the measurement marks on the bottle Do not use any other vessel to measure the water
?. [Tris Buffer pH 8.5 100mM]
Add 12.114 g of Tris Base
?. [Tris Buffer pH 8.5 100mM]
Mix thoroughly
?. [Tris Buffer pH 8.5 100mM]
Adjust to 1000 mL with ultrapure (type 1, >18 MOhms) water
?. [Tris Buffer pH 8.5 100mM]
Add 3.0 mL of Hydrochloric acid (Sigma-Aldrich, H1758-100ML) and mix well
?. [Tris Buffer pH 8.5 100mM]
Calibrate pH meter
?. [Tris Buffer pH 8.5 100mM]
Press Calibrate on the pH meter
?. [Tris Buffer pH 8.5 100mM]
Wash the pH probe with ultrapure (type 1, >18 MOhms) water, wipe, submerge in buffer 1 (pH 10) and Press Read
?. [Tris Buffer pH 8.5 100mM]
Wait until it stabilizes and says second buffer
?. [Tris Buffer pH 8.5 100mM]
Wash the pH probe with ultrapure (type 1, >18 MOhms) water, wipe, submerge in buffer 2 (pH 7) and Press Read
?. [Tris Buffer pH 8.5 100mM]
If the calibration is successful, the screen should indicate CALIBRATED
?. [Tris Buffer pH 8.5 100mM]
Adjust to pH 8.5
?. [Tris Buffer pH 8.5 100mM]
Wash the pH probe with ultrapure (type 1, >18 MOhms), wipe, submerge in the Tris solution
?. [Tris Buffer pH 8.5 100mM]
Press read and wait for the reading to stabilize
?. [Tris Buffer pH 8.5 100mM]
Acceptable pH values range between 8.4 to 8.6, if pH value is in that range, the solution is ready
?. [Tris Buffer pH 8.5 100mM]
If the pH is above 8.6 add 100 µL of HCl
?. [Tris Buffer pH 8.5 100mM]
If HCl has been added measure the pH againReuse the same Nalgene bottle to make new Tris Buffer pH 8.5 100mM until the initial expiring date (
?. [PBS low barium 1x]
Use a new 1 L Nalgene bottle, label with Brady lab printer (font 14pt, bold, banner landscape, 1/2" tape)  as follow:     First line: PBS low barium 1x    Second line: Exp.: MM/DD/YY  (expiration date: add 1 month to the preparation date in a new bottle)
?. [PBS low barium 1x]
Add 900 mL of ultrapure (type 1, >18 MOhms) water in 1 L Nalgene bottle Use the measurement marks on the bottle Do not use any other vessel to measure the water
?. [PBS low barium 1x]
Use 50 mL disposable Falcon tube to measure  (2x50 mL) of PBS 10x Low Barium stock solution
100 mL
?. [PBS low barium 1x]
Mix thoroughly
?. [PBS low barium 1x]
Reuse the same Nalgene bottle to make new PBS low Barium until the initial expiring date (
?. [Gelatin 10% stock, 100x]
Warm up  Ultrapure Water (Invitrogen, 10977-015) in a 50 mL
20 mL