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, P087.1) for 3 days at 4 °C. Do not cut lobes into smaller pieces

, Store tissue sections in reagent B at 4 °C. In this reagent, liver tissue can be stored up to 6 months

V. Vibratome and W. Leica, Germany) as follows: 3.1. Fix the liver lobe in the specimen holder using glue (Histoacryl ® Gewebekleber, B.Braun GmbH, Melsungen, Germany, 9381104) and wait for 2 min, Immediately before staining, slice the liver lobe parallel to its surface using a vibrating blade microtome

, Fix the vibratome feather blades (blades VT, Leica Microsystems, Wetzlar, Germany, 14020542056) tightly into the knife holder and install into the vibratome. Press the start button

, Remove tissue sections from reagent A and immerse in heated reagent C (in the 24-well tissue culture plate) for 2 min

, During the 2-min incubation, reheat reagent C (left in the plastic jar) again for 2 min and repeat steps 2.2. and 2.3. for an additional 9 times

, Cool tissue sections for 20 min at room temperature

, Wash tissue sections three times for 10 min each in reagent A at room temperature

, Blocking serum step: 4.1 Pipette 1 ml of reagent E into each well of a 24-well tissue culture plate

, Remove tissue sections from reagent A and immerse in reagent E

, Incubate tissue sections in reagent E for 2 h at room temperature in a 24-well tissue culture plate

, G2781, 1:2,000) and goat anti-mouse DPPIV/CD26 ectodomain (R&D systems, Primary antibodies: 5.1 Dilute rabbit anti-glutamine synthetase

, Pipette 1 ml of the diluted antibodies (from step 5.1.) into each well of a 24-well tissue culture plate

, Remove tissue sections from reagent E and immerse in the diluted antibodies

, Wash tissue sections three times for 10 min each in reagent A at room temperature

, :200) and alexafluor ® 647-conjugated AffiniPure F(ab')2 fragment donkey anti-mouse (DMs, Dianova GmbH, Secondary antibodies: 7.1 Dilute alexafluor ® 488-conjugated AffiniPure F(ab')2 fragment donkey anti-goat IgG (H + L) (Dianova GmbH, vol.1, pp.715-606

, Pipette 1 ml of the diluted antibodies (from step 7.1.) into each well of a 24-well tissue culture plate. 7.3 Remove tissue sections from reagent A and immerse in the diluted antibodies

, Wash tissue sections three times for 10 min each in reagent A at room temperature

, Prepare DAPI (4?,6-diamidin-2-phenylindol) solution as follows: To 10 ml of distilled water, add 1 µl of DAPI (Invitrogen, Darmstadt, Germany, D3571) and mix well, Counterstaining: 9

, Pipette 1 ml of the diluted DAPI (from step 9.1.) into each well of a 24-well tissue culture plate

, Remove tissue sections from reagent A and immerse in the diluted DAPI

, Incubate tissue sections with DAPI for 90 min at room temperature

, Wash tissue section three times for 10 min each in reagent A at room temperature

, Wash tissue sections for 10 min in distilled water at room temperature

, No. J1800AMNZ) using FluorPreserve reagent (Merck; Cat. No. 345787), cover with microscope cover glass (Thermo scientific, Gerhard Menzel GmbH

, Protocol for S-phase visualization (example of a result: with DAPI. Samples: 75-100-µm vibratome sections kept in reagent

, Wash tissue sections three times for 10 min each in reagent A at room temperature

, Antigen de-masking: 2.1. Pipette 1.25 ml of reagent C into a 2-ml Eppendorf tube

, Bovenden, Germany) at 95 °C for 25 min with shaking, Cook tissue sections in reagent C using a ThermoMixer (Model HTM 130L, HLC Biotech

, Cool tissue sections for 20 min at room temperature

, Wash tissue sections three times for 10 min each in reagent A at room temperature

, HCl digestion: 4.1. Pipette 2 ml of reagent D into each well of a 24-well tissue culture plate, p.83, 1836.

. , Remove tissue sections from reagent A and immerse in reagent D

, Incubate tissue sections in reagent D for 10 min at room temperature

, Wash tissue sections three times for 10 min each in reagent A at room temperature

, Blocking serum step: 6.1. Pipette 1 ml of reagent E into each well of a 24-well tissue culture plate

. , Remove tissue sections from reagent A and immerse in reagent E

, Incubate tissue sections in reagent E for 2 h at room temperature in a 24-well tissue culture plate

, :500) in reagent F in a 15-ml tube, First Primary antibody: 7.1. Dilute rat anti-BrdU, clone BU1/75 (ICR1) (AbD SEROTEC

, Pipette 1 ml of the diluted antibodies (from step 7.1.) into each well of a 24-well tissue culture plate

, Remove tissue sections from reagent E and immerse in the diluted antibodies

, Wash tissue sections three times for 10 min each in reagent A at room temperature

, First secondary antibody: 9.1 Dilute alexafluor ® 488-conjugated AffiniPure F(ab')2 fragment donkey anti-rat IgG (H + L) (Dianova GmbH, vol.1, pp.712-546

, Pipette 1 ml of the diluted antibodies (from step 9.1.) into each well of a 24-well tissue culture plate

, Remove tissue sections from reagent E and immerse in the diluted antibodies

, :2,000) in reagent F in a 15-ml tube, 11.1 Dilute rabbit anti-glutamine synthetase

, Pipette 1 ml of the diluted antibodies (from step 11.1.) into each well of a 24-well tissue culture plate

, Remove tissue sections from reagent E and immerse in the diluted antibodies

, Wash tissue sections three times for 10 min each in reagent A at room temperature

, :200) and alexafluor ® 647-conjugated AffiniPure F(ab')2 fragment donkey anti-mouse (DMs, Dianova GmbH, Second secondary antibodies: 13.1 Dilute Cy?3-conjugated AffiniPure F(ab')2 fragment donkey anti-rabbit (Dianova GmbH, vol.1, pp.715-606

, Pipette 1 ml of the diluted antibodies (from step 13.1.) into each well of a 24-well tissue culture plate

, Remove tissue sections from reagent E and immerse in the diluted antibodies

, Wash tissue sections three times for 10 min each in reagent A at room temperature. 15. Counterstaining: 15.1 Prepare DAPI (4?,6-diamidin-2-phenylindol) solution as follows: To 10 ml of distilled water, add 1 µl of DAPI

, Pipette 1 ml of the diluted DAPI (from step 15.1.) into each well of a 24-well tissue culture plate

, Remove tissue sections from reagent A and immerse in the diluted DAPI

, Incubate tissue sections with DAPI for 90 min at room temperature

, Wash tissue section three times for 10 min each in reagent A at room temperature

, Wash tissue sections for 10 min in distilled water at room temperature

, No. J1800AMNZ) using FluorPreserve reagent (Merck; Cat. No. 345787), cover with microscope cover glass (Thermo scientific, Gerhard Menzel GmbH

, During slicing, discard the first and last tissue sections to avoid the margin of a tissue block where the structure often appears altered

, All incubation and washing steps should be done on a shaker (Type KL2, Edmund Buhler GmbH

, Prepare all antibodies and DAPI directly prior to use

, 706) or in a 15-ml tube (SARSTEDT, Numbrecht, Germany, 62.554.502) or a 50-ml tube, Prepare antibodies, buffers and DAPI in 0.5-ml Eppendorf, vol.72

, Incubate tissue sections overnight in 24-well tissue culture plates

C. Kcl and K. , Germany, Art.-Nr. 6781.1), 10 g of potassium dihydrogen phosphate, 10× PBS: 1.1 To 4 l of distilled water, add 10 g of potassium chloride, vol.3904

, Bring the volume to 5 l with distilled water

, For 1 l of 1× PBS: 2.1 To 900 ml of distilled water, add 100 ml of prepared 10× PBS and mix well

, To 1,000 ml of reagent A, add 300 g of glucose (D-(+)-glucose anhydrous, Nr. X997.2) and mix well

, To 100 ml of the prepared glucose (in step 1), add 100 ml of

C. Reagent,

, To 800 ml of distilled water, add 2.1 g of citric acid monohydrate

D. Reagent, DNA denaturation agent)

C. , K. Germany, and A. Nr, NO76.1) as follows: To 10 ml of distilled water, add 10 ml of HCl and mix well, Prepare 2N hydrochloric acid

, To 97 ml of reagent A, add 3 g of bovine albumin fraction V, protease free (BSA, Serva Electrophoresis GmbH, Heidelberg, Germany, 11926, 100 g) and mix well

, 500 ml) and mix well

, To 97 ml of 1× PBS, add 0.3 g of bovine albumin fraction V, protease free (BSA, Serva Electrophoresis GmbH, Heidelberg, Germany, 11926, 100 g) and mix well

, 500 ml) and mix well

, Samples: 5-8 µm paraffin-embedded sections

, De-paraffinize the sections in four washes of roti-histol

. Karlsruhe-germany, 6640) for 5 min each

, Rehydrate the sections using descending ethanol

. Karlsruhe-germany,

, 9065.4) gradients (100, 95, 90, 70, 50 and 30 % ethanol, for 5 min each)

, Unmask the antigen of interest using an acid and heat treatment as follows

, Place the sections in a container, cover with freshly prepared reagent C and heat at 95 °C for 7 min in a microwave oven

, Top off with reagent C and reheat at 95 °C for 7 min

, Allow the sections to cool in the heated reagent C for approximately 20 min

, Wash the sections in reagent A three times for 5 min each

, Block the endogenous peroxidase by a methanolic hydrogen peroxide (H 2 O 2 ) treatment. For 100 ml of 30 % H 2 O 2 (Carl-ROTH; Karlsruhe-Germany; Art. No. 8070.4), add 100 ml of methanol

. Griesheim-germany, JT9093-3) and incubate the sections in the methanolic H 2 O 2 for 30 min at room temperature in a dark box

, Wash the sections in reagent A three times for 5 min each

, Block any unspecific binding using reagent E. Remove the sections from reagent A and immerse in reagent E. Incubate the sections in reagent E for 1 h at room temperature in a humified chamber

, Drain the blocking solution from the slides and apply two drops of avidin solution (Avidin biotin blocking kit; Vector Laboratories

, SP2001) for 15 min

, Remove the avidin solution from the slides and apply two drops of biotin solution (Avidin biotin blocking kit; Vector Laboratories

, Drain the biotin solution from slides. 12. Apply approximately 200 µl of the diluted primary antibody (in reagent F) at the recommended concentration per slide

, Wash the sections in reagent A three times for 5 min each

, Apply approximately 200 µl of the diluted secondary biotinylated antibody (in reagent F) at the recommended concentration per slide (supplemental table 1B) and incubate the sections for 60 min

, Wash the sections in reagent A three times for 5 min each

, Apply approximately 200 µl of the diluted horseradish peroxidase (in reagent F) at the recommended concentration per slide (Supplemental Table 1B) and incubate the sections for 60 min

, Wash the sections in reagent A three times for 5 min each

, Prepare the 3,3?-diaminobenzidine (DAB) peroxidase substrate (Vector Laboratories; Dossenheim-Germany

, To 5.0 ml of distilled water, add 2 drops of the buffer stock solution and mix well

, Add 4 drops of the DAB stock solution and mix well

, Add 2 drops of the hydrogen peroxide solution and mix well

, Incubate the sections with the substrate working solution at room temperature for 4 min

, Wash the sections once in distilled water for 5 min

, Counterstain the sections in Mayer's haematoxylin (Merck, Langenfeld-Germany; art. Number: 1092492500) for 120 s and immediately wash the sections under tap water for 10 min

, Dehydrate the sections using ascending ethanol

. Karlsruhe-germany,

, 9065.4) gradients (30, 50, 70, 90, 95 and 100 % ethanol, for 10 s each)

, Wash the sections twice in roti-histol

. Karlsruhe-germany,

, 6640) for 3 min each

, 1079600500), cover with microscope cover glass (Thermo scientific, No.J1800AMNZ) using Entellan (Merck-Millipore

, Cover the tissue slice with one drop of Olympus immersion oil type-F

, Prepare the z-stacks by 20× or 60× oil objectives (the settings are described in supplemental table 2)

, Adjust the parameters of the scanning mode as described in supplemental table 2

, For architectural staining, select DAPI (blue; nuclear staining), alexafluor 488 (green; DPPIV/CD26 signal), Cy3 (white; GS staining) and Alexafluor 647

, Stop the pre-scan mode and activate the 3D scanning mode

, Define the scanable first and the last z-levels

, Press the 'start' button to start manual recording, adjust the laser energy settings (HV, gain and offset) to approximately every 1 µm of z-level and record each level

, Press the 'end' button and start the automatic recording mode

, Deconvolute the 3D image using AutoQuant X3 (Bitplane). The 3D image is then ready for the pre-processing steps as described in the 'Image Processing with TiQuant' section

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