Biochemical and immunohistological characterization of brain tissue

Free-floating staining 

Sections (30 µm thick, cut with cryostat) are in PBS at +4°C in 6 well plates (when recently cut, up to 2-3 weeks), or in storing solution (for long-term storage) in 2mL Eppendorf tubes (at -20°C).

1. Transfer selected sections in 24 well plate with 1ml PBS/well using a small brush
2. [Wash briefly sections with PBS-T if stored in storing solution)]
3. OPTIONAL: Antigen retrieval (if necessary for antibody binding, otherwise skip this step) 
   -    Pre-warm 2 mL Eppendorf tubes filled with 10 mM citrate buffer pH 6 (dilute in water from 1 M stock solution) in a heat block at 96°C (around 30 min)
   -    Bring the slices in the pre-warmed citrate buffer and put them back in heat block  for around 20 min (shaking slowly so that slices do not stick to each other)
   -    Wash 3x PBS-T, 10 min., Room Temperature (RT), shaker
4. Permeabilization in PBS-T for 30 min at RT (shaker) 
5. Blocking for 1h at RT in blocking solution (shaker)
6. Incubation with primary antibodies (use apropiate dilution) in blocking solution (400-500µL/well) overnight (O/N) at +4°C (shaker)
7. Washes with PBS-T, 3x10min (shaker)
8. Secondary antibody incubation (dilution 1:500-1:1000) + Hoechst (1:2000) in blocking solution for 2h, RT, shaker (from here on keep sections in the dark)
9. Washes with PBS-T, 3x10min (shaker)
10. OPTIONAL: Thiazine-red staining (always after secondary Ab: 2µM Thiazine red (final concentration) in PBS for 20 min 
11. 3x10min PBS-T washes (shaker)
12. Bring sections on a 10 cm dish with PBS and some PBS-T (makes sections easier to mount) and mount sections using a brush on glass slides. Let sections dry on the slide (around 30 min-1h). Then add mounting medium and put coverslips by gently and evenly pressing. Leave slides O/N at RT (in the darkness) and then store at +4°C

NOTES: 

• When using a brush to transfer slices from one solution to another, especially when doing different stainings in paralel, be aware of cleaning the brush in between to avoid cross contamination of antibodies.

• Do not use treated glass slides since permafrost could give some troubles to mount sections. 

• Mouse perfusion is a very important step which tremendously affects the quality of the staining (recommendation: transcardially perfuse with cold 0.1M PBS for 5 min followed by 4% paraformaldehyde (PFA) in 0.1 M PBS for 10-15 min. Check that body is stiff, specially neck. Carefully isolate brains and postfixed for 20 min in 4% PFA in 0.1 M PBS. Then briefly wash the brains with PBS and transfer them to 30% sucrose in 0.1 M PBS for cryopreservation. When brains sink in the tube, embed them in optimal cutting temperature compound (Tissue-Tek O.C.T., Sakura), freeze them on dry ice and store at -80°C until sectioning.

Reagents and buffers:

• PBS-T: PBS with 0.5% Triton
• Blocking solution: 5% Normal Goat/Horse Serum in PBS-T (use species according to secondary antibody)
• Hoechst 33342 (H3570, ThermoFisher): Alternatively one can use DAPI
• Thiazine Red (27419.123, VWR)
• Glass slides (Menzel AAAA000001##12E, Thermo Scientific)
• Mounting medium (Flouromount, F4680-25ml, Sigma)
   
• Storing Solution for floating sections:

 For 500ml:  Glycerol :            150ml

                     Ethylenglycol:     150ml

                     H₂O bidest:         150ml

                    PO₄ Buffer 10x:  50ml

      PO₄ Buffer 10X, 0,25M, pH 7,2-7,4:

      NaH₂PO₄ xH₂O: 65gr

      NaOH: 15gr

      5N HCL:   ca. 2ml   (pH 7,2-7,4)

      Fill till 400ml with water and stir

• 1 M citric acid buffer pH 6 stock solution (for antigen retrieval)
  For 250mL
• Citric Acid monohydrate  (Mr 210.14 g/mol): 52,53g
• Dissolve in 150 mL H₂O
• Add additional 30 mL H₂O, adjust pH to 6 by adding 10 M NaOH solution (cca. 60 mL)
• After adition of 10 M NaOH, pH is typically around 5, one needs to add 10-20 NaOH pellets (let it cool down) to bring pH to 6. 
• When pH 6 is reached sterile filter

• Use for antigen retrieval 10 mM sodium citrate, pH 6 (dilute stock 100X with H₂O, not necessary to adjust pH again but check with lackmuspapier)
  For 10 M NaOH solution:
  (NaOH Mr 40g/mol)
  For 100mL: dissolve 40g NaOH in 100 mL dH₂O (exotermic reaction, add NaOH slowly)

   

BIOCHEMICAL ANALYSIS OF Aβ FROM MOUSE BRAIN 

Protocol for brain homogenization and fractionation according to protein solubility

This protocol includes a buffer-dependent extraction of proteins according to protein solubility in each buffer: DEA extraction (soluble proteins) followed by RIPA extraction (membrane, nuclei, mitochondrial and cytosolic proteins) and FA extraction (insoluble proteins). Protocol is modified from Willem et al., Nature, Vol.526 (2015) pp443.

Instruments and reagents:

• Precellys machine: Cat#: P000669-PR240-A, Bertin Instruments

• Precellys tubes (Tissue homogenizing CKMix - 2mL): Cat#: P000918-LYSKO-A, Bertin Corp.

• 1.5 mL Eppendorf tubes

• Protease Inhibitor Cocktail (PI mix; 500 x): Cat#: P8340, Sigma-Aldrich

• Water bath sonicator

• Ultracentrifuge and 1.5 mL ultracentrifuge tubes

• Tabletop centrifuge (cooled)
   

Buffers:

• 1X DEA Buffer : 0,2% Diethylamine (Cat#: 471216, Sigma-Aldrich) in 50 mM NaCl, pH 10.
• 1X RIPA Buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate (add Na2EDTA freshly before use)
• 0.5M Tris, pH 6.8
• 1M Tris pH 9.5
• FA solution: 70% Formic acid (in water)

This protocol is adjusted to the initial amount of ~200 mg of tissue (1 brain hemisphere). 

1. Brain hemispheres were stored after isolation at -80°C. Once taken from -80°C freezer, place them on dry ice to keep frozen (homogeneization can be also done right after brain isolation).
2. In Precellys tubes, add 1mL (min. volume for 1 hemisphere) of DEA buffer + protease inhibitor (PI) mix (1:500 from the 500X stock) per tube and place on ice (on pre-cooled metal holder). Add one frozen hemisphere in each Precellys tube containing DEA buffer + PI mix (check carefully that all tubes are properly closed otherwise you risk to loose material during shaking).
3. Homogenize using Precellys machine at 6500 rpm, 30 sec, 8°C (placed in a cold room).
4. Centrifuge for 10 min at 5000g to pellet membranes, nuclei and mitochondria (this pellet will be used further for RIPA extraction).
5. Transfer 0,60 – 0,75 mL supernatant carefully from Precellys tubes in ultracentrifuge 1,5 mL tubes (check with first sample how much you can take without disturbing the pellet, then take the same amount from each sample; remove the excess foam and liquid from the remaining pellets).
6. Ultracentrifuge the supernatant 30 min at 100 000g (4°C) (while this is ongoing go to step 9 to work with the pellet from steps 5-6).
7. Collect the supernatant (DEA fraction) from the ultracentrifuge tube: (first check how much volume you can take from the first tube without disturbing the pellet:  ~0,5 – 0,6 mL), then add 10% of 0,5 M Tris, pH 6,8 to each tube containing DEA fraction to adjust pH = DEA fraction. Store at -80°C until use.To the little pellet within the ultracentrifuge tube add 0,1 mL RIPA buffer (1x + PI mix) and resuspend by shaking briefly à go to step 11 to continue with RIPA extraction.
8. Resuspend the pellet from step 5 in 1 mL RIPA (+PI mix) (within the Precellys tubes) by shaking briefly and homogenize it with Precellys at 5000 rpm for 12 seconds at 8°C. 
9. Centrifuge 10 min at 5000g (6000 rpm in cooled tabletop centrifuge) to remove insoluble material (pellet for FA extraction, step 12).
10. Take the supernatant from step 10 and combine with the resuspenend ultracentrifugation pellet from step 8 (in 0,1 mL RIPA buffer) and ultracentrifuge for 60 min 100000g (4°C) and collect supernatant (discard pellet) = RIPA fraction (store at -80°C until use). 
11. Resuspend the pellet from step 10 in 0,5 mL (up to 1 mL depending on the predicted Aβ amount) 70% formic acid (FA + PI mix) by briefly shaking and sonicate for 7 min at 4°C
12. Centrifuge for 30 min 20000g (14 000 rpm, tabletop centrifuge, 4°C) and collect supernatant without disturbing and avoiding to take the “lipidic” layer in the surface of the tube = FA fraction (dilute 1:20 in 1M Tris pH 9.5 and store at -80°C until use).

Proteins from DEA and RIPA fractions can be quantified by protein quantification methods like BCA assay. Insoluble proteins from FA fraction cannot be quantified with such methods, therefore sample loading for western blotting is done according to initial brain tissue weight (before homogenization) or to the protein concentrations of samples from DEA fraction (since it is the least manipulated faction). 

Western blotting for soluble and insoluble Aβ 

1. Sample preparation: For Aβ detection, 20-30 µg of DEA or RIPA, or up to 20 µL of FA faction brain homogenates are combined with 4x loading buffer (final concentration 1x, such as Laemmli buffer, that contains a reducing agent like β-mercaptoethanol), heated for 5 min at 95°C and loaded into Tris-Tricine gels (Novex™ 10-20 % Tricin-Protein-Gels, EC66252BOX, Thermo Fisher Scientific) in 1x Novex™ Tricin SDS-Running buffer (LC1675, Thermo Fisher Scientific) using the XCell SureLock Mini-Cell Electrophoresis System (EI0001, Thermo Fisher Scientific).

2. Transfer proteins to nitrocellulose membranes (0.1 mm, GE Healthcare) in 1x Tris/Glycine Buffer (1610771EDU, BioRad) and boil the membranes for 5 min in PBS before blocking (this step can also be done using a microwave). 

3. Block for 1h in blocking solution containing 0.2% I-Block (Thermo Fisher Scientific) and 0.1% Tween 20 (Merck) in PBS.

4. Primary antibody incubation (for detecting human Aβ use  MABN2273 (Merck Millipore) at a concentration of 1 mg/mL overnight at 4°C in 0.2% I-Block (Thermo Fisher Scientific) and 0.1% Tween 20 (Merck) in PBS.

5. Wash 3x with PBS-T (1X PBS, 0.1% Tween) for 10 min.

6. Secondary antibody incubation: 1h with corresponding anti-HRP conjugated secondary antibody (1:4000) at room temperature. 

7. Wash 3x with PBS-T (1X PBS, 0.1% Tween) for 10 min.

8. Antibody detection (membrane development) is done using chemiluminescence detection reagent ECL (Thermo Fisher Scientific) according to manufacturer instructions.

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