Isolation of Mononuclear Cells from solid tumors
Tumor infiltrates isolation modified for colon
B16-OVA tumor induction
Immunohistochemistry Stainings (IHC)

Arginase 1

FoxP3 / IL-17

CD3

CD8a

CD163

ERG

F4/80

F4/80 - P-STAT3 double staining

Gr1

PTEN

Isolation of Mononuclear Cells from solid tumors

Natascha 12.12.09 modified after: T helper 17 cells promote cytotoxic T cell activation in tumor immunity.

Martin-Orozco N, Muranski P, Chung Y, Yang XO, Yamazaki T, Lu S, Hwu P, Restifo NP, Overwijk WW, Dong C. Immunity. 2009 Nov 20;31(5):787-98; and (Vremec et al., 2000).

Exp:… ……… /………………..……EP……

Need: camera, scissors and pinzette

Petri-dishes, PBS with 2% FCS

Ruler, Nunc-vial, dry-ice

Isolate draining (inguinal) and spleen per mouse

Take lungs in 2ml 10% Formalin, take blood for serum, take prostate/tumor (some for -80°C)

different FACS analysis due to multi-color staining on Fortessa, spleen - Calibur

Mice: Body weight of the mice in RN

Tumor weight in Lab

mouse weight +Seminal V -prostate

1. Measure tumor volume-

prepare tumor from killed mice- kill mice individually take a picture with a ruler!

+/- Seminal vesicles -> all together

2. Cell isolation from tumor

place the tumor in a 6-well plate from the petri-dish containing ice-cold PBS + 2% FCS

Cut the tumor into small pieces (razor blade or small knifes) in a 6 well plate

digest with 1 mg/ml collagenase D for 45 min at 37°C (5 ml / tissue)

number of mice: 1 5 ml

For 10ml: take 8.55 ml PBS 4,1 ml

+ 100µl Collagenase (250mg/ml) / (100mg/ml) 50µl / 125µl

+ 100µl DNase (100mg/ml) / (20mg/ml) 50µl / 250µl

+ 1ml MgCl (for DNase activity 25mM) 500µl

+ 200µl Serum 100µl

pre-warm at 37°C

5 min at 37°C with 0.01 EDTA for prevention of DC-T cell aggregates

+ 100µl EDTA (0.5M) per 5ml

put through a 70 µm cell strainer placed in a 6 well dish (back of 1 ml syringe plunger)

Collect the single cell suspension into a 15 ml tube on ice

Wash the cell-strainer with PBS and add to the tube until the solution is clear.

Take small aliquot (250µl) of digested tumor cells for (cell counting) surface staining (CD4 PE, CD8 APC, CD45 FITC, DX-5 PE-Cy7).

Centrifuge for 8-10 min at 1400rpm end: 1 TU samples

Meanwhile isolate draining LN and spleens with cell strainer

Prepare the Percoll 30% and 70% in PBS (+Serum) (Sigma P1644-500ML)!

20ml 30% Percoll: 6ml +14ml PBS: number of mice: 5ml x …1.. = …5……..ml

10ml 70% Percoll: 7ml + 3ml PBS: number of mice: 2,5ml x …1.. = …2,5……..ml

· Cells are resuspended in 30% Percoll: 5ml

o Turn the pipette boy to S (slow)

o Overlay very slowly with the lower (and lighter) 30% Percoll solution containing the cells onto the already provided 70% Percoll 2,5ml

o Centrifuge (1400rpm, 20min, RT , NO BRAKE)

o Remove the surface – tumor cell debris and fat with 5ml pipette or 1ml Tip

o Carefully suck off the interphase with mononuclear cells by using the pipette boy and get rid of Percoll contamination by washing

· Transfer into 15ml tubes, wash two times with PBS (+2%FCS)

· Centrifuge (1400rpm, 7min, 4°C)

Split cells: 1/3 for surface and 2/3 for IC (1/3 eBioscience [IC C], 1/3 Biolegend [IC D])

Start with restimulation!! (IC D)

Take 1/3 surface and split again – use 1/3 for Surface B (Calibur) and other 1/3 for Surface A (Fortessa), 1/3 for Surface M (Calibur)

2/3 of cells for IC (1/3 has to be restimulated for 4 hours – proceed on page 5), 1/3 for eBio TF Fortessa staining

Textfeld: Resuspend prostate
in 2,5 ml media (sum 7 tubes)
* 500µl/tube for IC (Fortessa)
* 280µl/tube for others

Tumor infiltrates isolation modified for colon

(Mario Kuttke)

Materials:

Icebox
Biorad Counting chambers
Eppi for re-genotyping
Scalpel
Scissors (sterile)
Forceps (sterile)
Petri dish
2x PFA for FACS
MACs beads stuff
50ml tubes (4x)
15ml tubes (1x)
2x Syringe+filter for sterile filtration of DNAse & Collagenase Mix

Reagents:

Percoll: Sigma P 4937
10x PBS, autoclaved
1x PBS, autoclaved, 110ml/mouse
BSA: Sigma A 9418
70µm strainer (734-0003 ZELL-SIEBE 70µm WEISS Corning B.V. / 352350)
RPMI for DNAse and Dissociation solution
Complete RPMI + (5 mM) EDTA [30ml/mouse]
à 3ml 0.5M EDTA in 300ml RPMI complete (+15 mg gentamycin + 3 ml PenStrep)

Dissociation medium (5ml per tumor):

o DNase I (Worthington Biochemicals LS 002139) 5 mg

o Collagenase P ( Roche 11249002001) 12.5mg

o Collagenase/Dispase (Roche 11097113001) 12.5mg

o RPMI to 5ml

o Filter sterilize 0.22µm

· DNASE SOLUTION (1ml per tumor)

o Can be aliquoted and stored at –20^oC. Make fresh solution is best!

o DNase I (Worthington Biochemicals LS 002139) 0.5 mg

o RPMI to 1 ml

o filter sterilize on 0.45 micron filter

· Percoll gradient (2x if you want to isolate IELs as well)

· Mix 9 vol Percoll with 1 vol PBS 10x = Percoll 100 4ml (3,6ml Percoll +0,4 ml 10xPBS)

· Make up Percoll 35 (35% Percoll 100 + 65% PBS) 4ml (1,4ml+2,6ml)

· Make up Percoll 60 (60% Percoll 100 + 40% PBS) 4ml (2,4ml+1,6ml)

· Prepare your gradients as follows:

· Add 4 ml Percoll 35 to a 15 ml tube and underlay with 4ml Percoll 60

Tumor associated macrophage isolation PROCEDURE:

Prepare fresh Digestion medium and DNase Solution and keep it at room temperature (RT) during the procedure
Preheat incubator and EDTA/RPMI to 37°C
Collect the colon in a 50 ml tube containing 10 ml PBS and bring it under the tissue culture hood.
Aspirate the PBS. Cut the colon longitudinally with scissors and chop into small pieces (into a fine mush) using scissors on the lid of a 50ml tube (or a scalpel on an upturned petri dish). Put pieces from individual colon in 50 ml Falcon tubes and fill up to 10 ml with PBS
Invert several times, allow to sediment and discard supernatant (aspirate off)
Add Pre-warmed RPMI/EDTA up to 10 ml and incubate 15 minutes @ 37C shaking (shake ~200 rpm), alternatively: incubate in cell incubator and shake every 5 minutes
Discard supernatant (SN) (or collect in separate tube if you want to isolate IELs), repeat twice (pool with 1^st SNs for IELs> spin down, wash> put on gradient (see below))
Add PBS to ~15 ml, invert and incubate for 10 minutes at RT, discard SN (take care to remove everything- residual EDTA inhibits the collagenase!) and add 5 ml of Digestion medium in a 50 ml tube.
Incubate 20-60 minutes, 37^oC shaking incubator. During this time, prepare percoll gradiants (60%/35%). 4ml 60% percoll with 4ml 35% percoll overlay in a 15ml tube.
Cool centrifuge to 4°C
Dissociate the tumor with a 10 ml pipette.
Pass the cell suspension through a 70 µm strainer (there will be some chunks remaining in the filter – leave them) in a new 50ml tube. Wash the strainer with 10 ml of PBS.
Spin 5 minutes, 300x g, +4^oC, BRAKES ON.
Aspirate the supernatant. Resuspend the cells first by taping the bottom tube with your finger and second by adding 1ml of fresh DNAse Solution per tube (resuspension is extremely important). Add 4 mls of PBS per tube. SLOWLY lay over the cell suspension on the Percoll gradient using a 5 ml pipette.
Spin for 20 minutes, 2000xg, +4^oC, BRAKES OFF.
Harvest the cells.

a. Aspirate the cells that are located between the 5ml suspension medium and 35% percoll. Usually this cell layer comes easily as one sheet.

b. With a transfer plastic pipette, harvest the tumor cells / macrophages that settled on the 60% Percoll. Try to remove all the cells, put them in a 50 ml tube, fill up with PBS.

Spin 5 minutes, 300x g, +4^oC, BRAKES ON.

B16-OVA tumor induction

(Julia Pisoni)

http://www.lgcstandards-atcc.org/products/all/CRL6475.aspx?geo_country=at

B16-OVA Tumormodell zur Ermittlung der Compound-Wirkung auf die Anti-Tumor Immunität:

WT-Mäuse werden, unter oraler Verabreichung der Compounds, mit B16 Zellen orthotopisch subcutan in die Flanke inokuliert. Dabei werden die Mäuse mit Isofluran betäubt und 1x10⁶ B16-Zellen werden in 200µl einer 1:2 PBS Matrigel Mischung mit einer Insulin Spritze (23 Gauge Kanüle) injiziert. Nach dem sichtbaren Anwachsen des Tumors (Tag 6) werden die Tiere 2x mit OVA-Antigen vakziniert (Tag 6, Tag 13) und das Wachstum bzw. die Abstossung des Tumors wird entsprechend dokumentiert. Der Versuch wird am Tag 21 beendet bzw. werden Mäuse aus ethischen Gründen vorher euthanasiert wenn der Tumor zu einer schweren Beeinträchtigung führt. Am Versuchsende wird der Tumor für die Histologie aufbereitet sowie die T-Zellen der Tumor Draining Lymphnodes mittels Durchflusszytometrie analysiert.

Exp.: 15.10.2013

è Thaw at least 1- 1,5 week prior to the injection and split at least two times

1x 10⁵ B16-OVA cells injected subcutaneously into de-haired (w) or shaved (m) left flanks of
8-12 week old female/male mice

1 *10⁵in 100 µl

How many mice:

1x: 100.000: ……8…mice… need …8*10⁵……… cells in ……800µl

EXTRA: 3x: 1.000.000: ……24………… need …2,4*10⁶……… cells in ……2400µl

1. Injection of B16- melanoma cells:

B16 –Ova- Melanoma cells: (modified after Christina 07.01.10)

Growth:

DMEM +++: Medium β-mercaptoethanol. (500ml + 2µl)

Split every second day (except weekend) 1:4 in relation to the area

Use cell culture dishes- not petri dishes- or cell culture flasks to grow in

Cells must be in their exponential growing phase for optimal results

DMEM +

· G418- BC (30.000U/ml) Biochrom: 80µl mikroliter pro 10ml.

· 10% FCS,

· Glutamat

· PenStrep

Harvesting:

Remove DMEM medium

Wash with PBS (2x)

Add 2-3 ml Trypsin (+EDTA Nr……) per flask (4 flasks for 18 mice)

Incubate for 3-5 min at 37°C

Add DMEM+++ medium (serum stops trypsin) and wash 2 times (12000rpm 7min)

(Zellen kleben stark, gut resuspendieren)

Freezing

5x10⁶ cells/ml

1:10 in DMSO FCS in the Mister cooler -80°C

Defrost fast – put into 37°, wash well with DMEM to get rid of the DMSO and put into a flask.

Injection:

Be careful not smaller than 0,4mm needle (27 gauge)

1x 10⁵cells in 100µl PBS mix gently every time you take out an aliquot- no air bubbles!

Use matrigel if tumor is subjected to IHC staining!

I.p or (depending on tumor model)

i.v. inject the mice- that have been below the red light for at least 15min.

measure tumor growth every 2^nd day with a caliper

Stainings

Immunohistochemistry Stainings (IHC)

Arginase 1 IHC Staining

- 5-10 min 60°C

- Rehydrate (10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest.

- Antigenretrieval steamer pH 6, 1h

- Let it cool off, then wash 3x in 1XPBST

- Block 10min in 3%H₂O₂ (in PBS)

- Wash 3x in 1XPBST

- 10min Avidin block

- Wash 3x in 1XPBST

- 10min. Biotin block

- Wash 3x in 1XPBST

- Surround tissue with DAKO pen

- 7min. Superblock (2 drops)

- Wash 3x in 1XPBST

- 1h mouseblock (2 drops)

- Wash 3x in 1XPBST

- 1° ab (Arg 1) 1h at RT / on at 4°C (1:500 in PBS 1% goat serum)

- Wash 3x in 1XPBST

- 10min. Biotinylated ab

- Wash 3x in 1XPBS (NO TWEEN)

- 10.min Streptav.-HRP

- Wash 3x in 1XPBS (NO TWEEN)

- AEC ( 20µl substrate +1000µl buffer)

- stop in tab water

- Wash in aqua dest.

- Hemalaun

- stop in tab water

- mount with Aquatex

sc-20150 Arginase I (H-52) rabbit polyclonal IgG Santa Cruz

FoxP3 / IL-17 double IHC Staining

- 30min-1h 60°C

- Rehydrate (3x10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest.

- Antigenretrieval steamer pH 9, 1h

- Let it cool off, then wash 3x in 1XPBS

- Block 10min in 3%H₂O₂ (in PBS)

- Wash 3x in 1XPBS

- 10min Avidin block

- Wash 3x in 1XPBS

- 10min. Biotin block

- Wash 3x in 1XPBS

- 7min. superblock

- Wash 3x in 1XPBS

- 1h mouseblock

- Wash 3x in 1XPBS

- 1° ab (FoxP3) 1h at RT (1:200 in PBS 1% BSA)

- Wash 3x in 1XPBS

- 10min. Biotinylated ab

- Wash 3x in 1XPBS

- 10.min Streptav.-HRP

- Wash 3x in 1XPBS

- AEC

- Wash in aqua dest., then 1XPBS

- Block 1h in 5% goat serum in PBS

- 2° ab (IL-17) 1h at RT or overnight (1:200 in PBS 1% BSA)

- Wash 3x in 1XPBS

- 30min. anti-rabbit biotinylated ab 1:100

- Wash 3x in 1XPBS

- 10.min Streptav.-HRP (ID labs)

- Wash 3x in 1XPBS

- AEC(20µl substrate + 1000µl Buffer)/ histogreen develop, stop in tap water

- Wash in Aqua dest.

- Hemalaun 1:5, 30s, stop in tap water

- Aquatex

Foxp3(F-9)

sc-166212 mouse mono

IL-17 Abcam ab91649

All washing steps in PBS-T (0.1%) until biotinyl. Ab, then only PBS!

Rabbit polyclonal: anti-rabbit biotinylated ab 1:100

DAB/Metal Concentrate

Thermo scientific # 34065 (=new cat. Number)

1:10

Histogreen

Linaris #E109

CD3 IHC Staining

- 5-10 min 60°C

- Rehydrate (10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest.

- Antigenretrieval steamer pH 6, 1h

- Let it cool off, then wash 3x in 1XPBST

- Block 10min in 3%H₂O₂ (in PBS)

- Wash 3x in 1XPBST

- 10min Avidin block

- Wash 3x in 1XPBST

- 10min. Biotin block

- Wash 3x in 1XPBST

- Surround tissue with DAKO pen

- 7min. Superblock

- Wash 3x in 1XPBST

- 1h mouseblock

- Wash 3x in 1XPBST

- 1° ab (CD3) 1h at RT / o n at 4°C (1:150-300 in PBS 1% goat serum)

- Wash 3x in 1XPBST

- 10min. Biotinylated ab

- Wash 3x in 1XPBS (NO TWEEN)

- 10.min Streptav.-HRP

- Wash 3x in 1XPBS (NO TWEEN)

- AEC ( 20µl substrate +1000µl buffer)

- stop in tab water

- Wash in aqua dest.

- Hemalaun

- stop in tab water

- mount with Aquatex

anti-CD3 (Early T Cell Marker), Clone: SP7, Thermo Scientific

CD8a IHC Staining

- 30min-1h 60°C

- Rehydrate (3x10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest.

- Antigenretrieval steamer pH9, 1h

- Let it cool off, then wash 3x in 1XPBST

- Block 10min in 3%H₂O₂ (in PBS)

- Wash 3x in 1XPBST

- 10min Avidin block

- Wash 3x in 1XPBST

- 10min. Biotin block

- Wash 3x in 1XPBST

- 7min. superblock

- Wash 3x in 1XPBST

- 1h mouseblock

- Wash 3x in 1XPBST

- 1° ab 1h at RT (1:100 in PBS 1% BSA)

- Wash 3x in 1XPBST

- 10min. Biotinylated ab

- Wash 3x in 1XPBS

- 10.min Streptav.-HRP

- Wash 3x in 1XPBS

- AEC (20µl substrate + 1000µl Buffer), develop, stop in tap water

- Wash in aqua dest.

- Hemalaun 1:5, 30s, stop in tap water

- Aquatex

All washing steps in PBS-T (0.1%) until biotinyl. Ab, then only PBS!

Rat-anti-mouse CD8a (53-6.7)

Biolegend cat# 100701

Stored at 4°C

CD163 IHC Staining

- 30min-1h 60°C

- Rehydrate (3x10min Xylen, 30s. 100%, 96%, 80%, 70%, 50%), A. dest.

- Antigenretrieval steamer pH9, 1h

- Let it cool off, then wash 3x in 1XPBS

- Block 10min in 3%H₂O₂ (in PBS)

- Wash 3x in 1XPBS

- 10min Avidin block

- Wash 3x in 1XPBS

- 10min. Biotin block

- Wash 3x in 1XPBS

- 7min. superblock

- Wash 3x in 1XPBS

- 1h mouseblock

- Wash 3x in 1XPBS

- 1° ab 1h at RT (1:200 in PBS 1% BSA)

- Wash 3x

- 10min. Biotinylated ab

- Wash

- 10.min Streptav.-HRP

- Wash

- AEC (20µl substrate + 1000µl Buffer), develop, stop in tap water

- Wash in aqua dest.

- Hemalaun 1:5, 30s, stop in tap water

- Aquatex

CD163 sc33560 (M-93)

Rabbit polyclonal IgG

All washing steps in PBS-T (0.1%) until biotinyl. Ab, then only PBS!

ERG IHC Staining

- 5-10 min 60°C

- Rehydrate (10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest.

- Antigenretrieval steamer pH 9, 1h

- Let it cool off, then wash 3x in 1XPBST

- Block 10min in 3%H₂O₂ (in PBS)

- Wash 3x in 1XPBST

- 10min Avidin block

- Wash 3x in 1XPBST

- 10min. Biotin block

- Wash 3x in 1XPBST

- Surround tissue with DAKO pen

- 7min. Superblock

- Wash 3x in 1XPBST

- 1h mouseblock

- Wash 3x in 1XPBST

- 1° ab (ERG EP111) 1h at RT / on (1:50eigentlich) in DAKO antibody diluent)

- Wash 3x in 1XPBST

- 10min. Biotinylated ab

- Wash 3x in 1XPBS (NO TWEEN)

- 10.min Streptav.-HRP

- Wash 3x in 1XPBS (NO TWEEN)

- AEC ( 20µl substrate +1000µl buffer)

- stop in tab water

- Wash in aqua dest.

- Hemalaun

- stop in tab water

- mount with Aquatex

F4/80 IHC Staining

- 30min-1h 60°C

- Rehydrate (3x10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), Aqua dest.

- Antigenretrieval microwave pH6 (3min. 800 W, 15 min 290 W)

- Let it cool off, then wash 3x in 1XPBS

- Block 10min in 3%H₂O₂ (in PBS)

- Wash 3x in 1XPBS

- 10min Avidin block

- Wash 3x in 1XPBS

- 10min. Biotin block

- Wash 3x in 1XPBS

- 7min. superblock

- Wash 3x in 1XPBS

- 1h mouseblock

- Wash 3x in 1XPBS

- 1° ab overnight (1:100)

- Wash 3x

- 10min. Biotinylated ab

- Wash

- 10.min Streptav.-HRP

- Wash

- AEC (20µl substrate + 1000µl Buffer), develop, stop in tap water

- Wash in aqua dest.

- Hemalaun 1:5, 30s, stop in tap water

- Aquatex

1° rat-anti-F4/80 1:100 in 1% goat serum; 150-200μl per slide

F480 macrophage monoclonal antibody

AbD Serotec

MCA497G

F4/80 staining (double staining with P-Stat3)

Bake paraffin embedded slides for 1h at 60°C
Rehydrate: 3x in Xylen, 10 min. each,; then 30sec. each in 100%, 96%, 80%, 70%, 50% EtOH; 5min in dH₂O
Antigenretrieval: 60min in steamer pH6 (Dako target retrieval solution, Citrate pH6 (10X) Ref.no. S2369
Wash 3 times in PBS-T
Block 10. min in 3% H₂O₂ in PBS
Wash 3x in PBS-T (if u like u can draw a line with a Dako pen on your slide to mark your tissue area)
Incubate 7. min with superblock
Wash 3 times in PBS-T
Block 1h in 5% goat serum in PBS
No washing, add 1° ab overnight at 4°C
Wash 3 times in PBS-T
2^nd ab 10min, wash 3 times in PBS-T
Streptavidin-HRP 10min (ID labs)
Wash with PBS
Develop with AEC Chromogen (drop Chromogen on slide, check under the microscope until the staining is complete 4-7min), stop in tap water, then in Aqua dest.
If no further ab staining: Hemalaun staining (1:5), coat with Aquatex

1° rat-anti-F4/80 1:100 in 1% goat serum; 150-200μl per slide

F480 macrophage monoclonal antibody

AbD Serotec

MCA497G

2° anti polyvalent biotinylated antibody (kit from ID labs contains also super block, mouse block) IDST1007 ?

The P-Stat3 staining done by Michi in AKH, with AP, not with HRP system!

P-Stat3 (Y705) Rabbit mAb (D3A7) Cell Signaling #9145L

PYStat3 staining

Bake paraffin embedded slides for 1h at 60°C
Rehydrate: 3x in Xylen, 10 min. each,; then 30sec. each in 100%, 96%, 80%, 70%, 50% EtOH; 5min in dH₂O
Antigenretrieval: 60min in steamer pH9 (Dako target retrieval solution, Tris/EDTA pH9 (10X)
Wash 3 times in PBS-T
Block 10. min in 3% H₂O₂ in PBS
Wash 3x in PBS-T (if u like u can draw a line with a Dako pen on your slide to mark your tissue area)
Incubate 7. min with superblock
Wash 3 times in PBS-T
Block 1h in 5% goat serum in PBS
No washing, add 1° ab overnight at 4°C
Wash 3 times in PBS-T
2^nd ab 10min, wash 3 times in PBS-T
Streptavidin-HRP 10min (ID labs)
Wash with PBS
Develop with AEC Chromogen (drop Chromogen on slide, check under the microscope until the staining is complete 4-7min), stop in tap water, then in Aqua dest.
If no further ab staining: Hemalaun staining (1:5), coat with Aquatex

2° anti polyvalent biotinylated antibody (kit from ID labs contains also super block, mouse block) IDSTM003

P-Stat3 (Y705) Rabbit mAb (D3A7) Cell Signaling #9145

F4/80+P-Stat3 for IF

Bake paraffin embedded slides for 1h at 60°C
Rehydrate: 3x in Xylen, 10 min. each,; then 30sec. each in 100%, 96%, 80%, 70%, 50% EtOH; 5min in dH₂O
Antigenretrieval: 60min in steamer pH6 (Dako target retrieval solution, Citrate pH6 (10X) Ref.no. S2369
Wash 3 times in PBS-T
Block 10. min in 3% H₂O₂ in PBS
Wash 3x in PBS-T (if u like u can draw a line with a Dako pen on your slide to mark your tissue area)
Incubate 7. min with superblock
Wash 3 times in PBS-T
Block 1h in 5% goat serum in PBS
No washing, add 1° antibody mix overnight at 4°C
Wash 3 times in PBS-T
2^nd antibody mix 30min, wash 3 times in PBS-T
Wash 3 times in PBS-T
DAPI (1:50.000) 5min.
Wash 3 times in PBS-T
Dako mounting media

1° Ab: 1° rat-anti-F4/80 1:100 in 1% goat serum; 150-200μl per slide

F480 macrophage monoclonal antibody

AbD Serotec

MCA497G

P-Stat3 (Y705) Rabbit mAb (D3A7) Cell Signaling #9145L

2° Ab: goat anti rat PE (invitrogen A10522) 1:200

goat anti rabbit FITC (invitrogen A11008) 1:200

Total Stat3 H190 (Santa Cruz): 1:80

F4/80+Total Stat3 for IF

Bake paraffin embedded slides for 1h at 60°C
Rehydrate: 3x in Xylen, 10 min. each,; then 30sec. each in 100%, 96%, 80%, 70%, 50% EtOH; 5min in dH₂O
Antigenretrieval: 60min in steamer pH6 (Dako target retrieval solution, Citrate pH6 (10X) Ref.no. S2369
Wash 3 times in PBS-T
Block 10. min in 3% H₂O₂ in PBS
Wash 3x in PBS-T (if u like u can draw a line with a Dako pen on your slide to mark your tissue area)
Incubate 7. min with superblock
Wash 3 times in PBS-T
Block 1h in 5% goat serum in PBS
No washing, add 1° antibody mix overnight at 4°C
Wash 3 times in PBS-T
2^nd antibody mix 30min, wash 3 times in PBS-T
Wash 3 times in PBS-T
DAPI (1:50.000) 5min.
Wash 3 times in PBS-T
Dako mounting media

1° Ab: 1° rat-anti-F4/80 1:100 in 1% goat serum; 150-200μl per slide

F480 macrophage monoclonal antibody

AbD Serotec

MCA497G

Total Stat3 H190 (Santa Cruz): 1:80 cat.# sc-7179 1:80

2° Ab: goat anti rat PE (invitrogen A10522) 1:200

goat anti rabbit FITC (invitrogen A11008) 1:200

Total Stat3

Bake paraffin embedded slides for 1h at 60°C
Rehydrate: 3x in Xylen, 10 min. each,; then 30sec. each in 100%, 96%, 80%, 70%, 50% EtOH; 5min in dH₂O
Antigen retrieval: 60min in steamer pH6 (Dako target retrieval solution, Citrate pH6 (10X) Ref.no. S2369
Wash 3 times in PBS
Block 10. min in 3% H₂O₂ in PBS
Wash 3x in PBS (draw a line with a Dako pen on your slide to mark your tissue area)
Incubate 7. min with superblock
Wash 3 times in PBS
Block 1h in mouseblock (ID lab)
Wash 3x, PBS
add 1° antibody total Stat3 1:80 in PBS+ 1% BSA overnight at 4°C
Wash 3 times in PBS
Biotinylated secondary ab 10min (ID labs)
Wash PBS 3x
HRP (ID labs) 10min
Wash 3x PBS
Develop with AEC 1ml buffer + 20 microL substrate (stop in tap water, then in Aqua dest.)
Hemalaun 1:5 (in Aqua dest.), 15-20 sec.

F4/80+Total Stat3

Bake paraffin embedded slides for 1h at 60°C
Rehydrate: 3x in Xylen, 10 min. each,; then 30sec. each in 100%, 96%, 80%, 70%, 50% EtOH; 5min in dH₂O
Antigen retrieval: 60min in steamer pH6 (Dako target retrieval solution, Citrate pH6 (10X) Ref.no. S2369
Wash 3 times in PBS
Block 10. min in 3% H₂O₂ in PBS
Wash 3x in PBS (draw a line with a Dako pen on your slide to mark your tissue area)
Incubate 7. min with superblock
Wash 3 times in PBS
Block 1h in mouseblock (ID lab)
Wash 3x, PBS
add 1° antibody F4/80 1:100 in PBS+ 1% BSA overnight at 4°C
Wash 3 times in PBS
Biotinylated secondary ab 10min (ID labs)
Wash PBS 3x
HRP (ID labs) 10min
Wash 3x PBS
Develop with AEC 1ml buffer + 20 microL substrate (stop in tap water), then in Aqua dest.
Block with 5% goat serum, RT
Do not wash, add. Total Stat3 ab (1:200 in PBS 1% BSA), 4°C ON
Wash 3x PBS
Biotinylated anti rabbit (vectastain) 30min (kit)
Wash
HRP (vectastain) 30min
Wash
DAB (Thermosc.) (Buffer 4°C, substrate -20°C 1:10)
Stop in tap water
Hemalaun 1:5 Merck in aqua dest. (Mayer), 15-20 sec (filter!).
Rinse in tap water
In aqua dest
Aquatex mounting media

F4/80 & Total Stat3

- 1h 60°C

- Rehydrate (3x10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), Aqua dest. 5min.

- Antigenretrieval autoclave 20min. pH6

- Let it cool off, then wash 3x in 1XPBS

- Block 10min in 3%H₂O₂ (in PBS)

- Wash 3x in 1XPBS

- 7min. superblock

- Wash 3x in 1XPBS

- 1h 5%goat serum in PBS

- 1° ab mix overnight 4°C

- Wash 3 times in PBS-T

- 2^nd antibody mix 30min, wash 3 times in PBS-T

- Wash 3 times in PBS-T

- DAPI (1:50.000) 5min.

- Wash 3 times in PBS-T

- Dako mounting media

1° Ab: 1° rat-anti-F4/80 1:100 in 1% goat serum; 150-200μl per slide

F480 macrophage monoclonal antibody

AbD Serotec

MCA497G

Total Stat3 H190 (Santa Cruz): 1:80 cat.# sc-7179 1:80

2° Ab: goat anti rat PE (invitrogen A10522) 1:200 this time 1:300

goat anti rabbit FITC (invitrogen A11008) 1:200

Gr1 IHC Staining

- 30min-1h 60°C

- Rehydrate (3x10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest.

- Antigenretrieval steamer pH9, 1h

- Let it cool off, then wash 3x in 1XPBS

- Block 10min in 3%H₂O₂ (in PBS)

- Wash 3x in 1XPBS

- 10min Avidin block

- Wash 3x in 1XPBS

- 10min. Biotin block

- Wash 3x in 1XPBS

- 7min. superblock

- Wash 3x in 1XPBS

- 1h mouseblock

- Wash 3x in 1XPBS

- 1° ab 1h at RT (1:200 in PBS 1% BSA)

- Wash 3x in 1XPBS

- 10min. Biotinylated ab

- Wash 3x in 1XPBS

- 10.min Streptav.-HRP

- Wash 3x in 1XPBS

- AEC (20µl substrate + 1000µl Buffer), develop, stop in tap water

- Wash in aqua dest.

- Hemalaun 1:5, 30s, stop in tap water

- Aquatex

Gr1 Rat anti mouse Ly6B.2

Serotec MCA771GA 1:200

iNOS mouse IgG

BD #610431 1:200

PTEN IHC Staining

- 5-10 min 60°C

- Rehydrate (5 min Xylen, 2 min. 96%, 96%, 1min 80%, 70% A. dest.

- Antigen retrieval (steamer pH 9, 1h)

- Let it cool off, then wash 3x in 1XPBST (0,1% TWEEN)

- Block 10min in 3%H₂O₂ (in PBS)

- Wash 3x in 1XPBST

- 10min Avidin block

- Wash 3x in 1XPBST

- 10min. Biotin block

- Wash 3x in 1XPBST

- Surround tissue with DAKO pen

- 7min. Superblock

- Wash 3x in 1XPBST

- 1° ab (PTEN) 1h at RT / on 4°C (1:200 in PBS 5% goat serum)

- Wash 3x in 1XPBST

- 10min. Biotinylated ab

- Wash 3x in 1XPBS (NO TWEEN)

- 10.min Streptav.-HRP

- Wash 3x in 1XPBS (NO TWEEN)

- AEC ( 20µl substrate +1000µl buffer)

- stop in tab water

- Wash in aqua dest.

- Hemalaun

- stop in tab water

- mount with Aquatex