Cy 3 GAD Mosaic

Immuno: rb∝GAD (1:40), labeled w/ Cy3⦁SAvidin (1:760, 90 min)
Tissue: 9/21/12, G1110C
Image: 10X, 9 panels, 1024x1024, 8 bit, 1 channel = 2747 X 2745 (cropped)=38x38" @ 72 dpi
Acquistion: 555 line, 2.0%, 1.58 Airy, Spd 7, Avg 2, Gain 559, Offset 0; 7.75 sec/panel
Tile: 15% overlap; Rotation not corrected (Zen could not stitch image)
Stitching: PS Photomerge, Auto mode
Processing: In PS, Assign & Convert to profile, Flatten and Crop. Colorized with Curves; Unsharp Mask: (Split: 64% Lighten, 31% Darken); Reduce size (from 9x9 to 6x6), convert to sRGB, save as JPEG
File Name: GAD 40k 10x 9 tile sm sRGB.jpg
Original File: 120921_Cy3GADA_40k_10X_9 tile_m0(...m8).tif

Random image of Cy 3 GAD in CP & overlying CTX. Captured for simple (fast imaging, limited tiles) source images to stitch. Conclusion: Zeiss Zen is capable of stitching images with 1 or fewer tiles...

Alexa 488⦁rb∝FITC

Immuno: rb∝Neurotensin (1:8k) labeled with Alexa 488⦁rb∝FITC (1:2K, Invitrogen)—to be used in double labeled series, in combination with a streptavidin conjugate. We also have Cy 2⦁ms∝FITC (1:2k, Jackson), which works just as good as the Alexa, and Dylight 488⦁gt∝FITC (Rockland), that's being tested now.
Image: 20 panels at 10x, 1024x1024 (x20), 8-bit
Acquisition: 488 line at 1.8%, 1.5 Airy, Gain 569, Offset 0, Speed 6, Avg 4 (31 sec/panel; 10m 30 sec total)
Tiling: 20% overlap; Rotation Correction 0.8087º (calibrated from Zeiss-provided macro from within Zen)--& the Zeiss software still wouldn't stitch
Stitching: PS 5 photomerge, auto mode.
Image Processing: In PS Assigned profile Prophoto and converted to Adobe '98. Flattened, Cropped and resized. Colorized with Curves. Sharpened with Unsharp Mask. Reduced size, converted to sRGB and saved a copy as a JPEG

Conclusion: Great staining, amplifies as much or more than streptavidin. However, 10x 1.5 Airy clearly doesn't capture all the data (when viewed at actual size of the original which is 14 x 11" at 300 dpi) and, unlike the NOS, would probably benefit greatly from acquiring in Z...

Neurotensin (1:8k) labeled with Alexa 488⦁rb∝FITC (1:2K)—to be used in double labeled series, in combination with a streptavidin conjugate.

DAPI Protocol

1. Order DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride). Available from Invitrogen as either D1306 (10 mg for $85.68) or D21490 (10 mg, ultrapure for $126.63)
2. Make stock solution of 5 mg/ml by adding 2 ml DI to one 10 mg vial. Stock solution = 14.3 mM. Note that sonication may be required to get it into solution
3. Stain. Incubate tissue in 300 nM DAPI for 1 - 5 min.
4. Rinse, coverslip, mount.

Note:

• 300 nM is a 1:47667 dilution of 14. 3 mM

which means

• 1 µl DAPI stock makes 47.7 ml DAPI working

which means

•  2 ml of stock will make 95.33 liters of DAPI stain

Dilution of Streptavidin Conjugates

Working Dilution of Streptavidin conjugates from Jackson Immuno is now standardized to a working concentration of 2 µg/ml, which is the upper end of Jackson's recommended 1 - 2 µg/ml.

Calculations are based on:

1. Each vial of streptavidin contains 1.0 mg, regardless of conjugate
2. Every conjugate is a different concentration (HRP at 1.0, Cy 2 at 1.2 and Cy 3/Cy 5 at 1.8 mg/ml)
3. Each concentration is reconstituted with a different volume of DI (1 ml, 950 µl and 650 µl, for HRP, Cy2 and Cy 3/Cy 5, respectively)

Calculations for 2 µg/ml:

 HRP⦁Streptavidin (1:500)

HRP⦁Streptavidin comes as 1.0 mg, rehydrated with 1.0 ml DI = 1 µg/µl. Therefore 2 µl's/ml are needed for a working concentration of 2 µg/ml. 2 µl's/ml = 1:500, or 20 µl per aliquot makes 10 ml at 1:500. 1 ml theoretically makes 50 aliquots at 20 µl per aliquot

 Cy 2⦁Streptavidin (1:526)

 Cy 2⦁Streptavidin comes as 1.0 mg, at a concentration of 1.2 mg/ml, and is rehydrated with 950 µl DI. 1 mg in 950 µl = 1.05 µg/µl and 2 µg = 1.9 µl. Therefore 1.9 µl's/ml are needed for a working concentration of 2 µg/ml, which would be 19 µl in 10 ml, which is a dilution of 1:526. Note that again 950 µl theoretically makes 50 aliquots at 19 µl per aliquot.

 Cy 3⦁Streptavidin (1:769)

 Cy 3⦁Streptavidin comes as 1.0 mg, at a concentration of 1.8 mg/ml, and is rehydrated with 650 µl DI. 1 mg in 650 µl = 1.54 µg/µl and 2 µg = 1.3 µl. Therefore 1.3 µl's/ml are needed for a working concentration of 2 µg/ml or 13 µl per aliquot for 10 ml, which is a final dilution of 1:769. Here, too, 650 µl theoretically makes 50 aliquots at 13 µl per aliquot

 Cy 5⦁Streptavidin and Alexa 647⦁Streptavidin (1:769)

 Cy 5⦁Streptavidin also comes as 1.0 mg, at a concentration of 1.8 mg/ml, and is rehydrated with 650 µl DI. 1 mg in 650 µl = 1.54 µg/µl and 2 µg = 1.3 µl. Therefore 1.3 µl's/ml are needed for a working concentration of 2 µg/ml or 13 µl per aliquot for 10 ml, which is a final dilution of 1:769. Here, too, 650 µl theoretically makes 50 aliquots at 13 µl per aliquot

IHC Test Results: 9/21/12

9/21/2012 Immuno Results (Preliminary)

General: Tissue in tatters, few sections per group, & many sections missing, but everything appeared to work and the sections were mounted in great anatomical register

Short vs Long: Seems clear that the short runs (2 hr in 2º, 90 min in SAvidin) worked as well as the long run (3+ hr in 2º, 2 hr in SAvidin). For the vGAT "blind control", where run conditions were not noted on either slide, I couldn't really tell the difference.

rb ∝ GAD (1: 20, 30 & 40k): All dilutions worked, but (I think) 1:20k was best--more because labeling was good and background was low, so there is little cost to going with 1:20k, rather than some particular gain

rb ∝ DYN (1: 5, 10 & 15k): Could see well-labeled fibers at all dilutions, but no clear concentration of fibers in a recognizable terminal field. Could see lots of labeled cells, somewhat concentration dependent so am not absolutely sure that this is specific labeling. To accurately assess, comparison with DAB series needed to identify the most prominent terminal fields, and the best sections/levels. If not present in these test sections, then the test series will need to be run again

rb ∝ vGAT (1: 2.5, 5 & 10k): Labeling saturated making it difficult to distinguish individual components at 1:2.5k, and it looked as though labeling may have fallen off a bit at 1:10k. Labeling at 1:5k looked great (although I'd bet that 1:7.5k would be optimal). As noted above, labeling group was not recorded on either slide.

Alexa 488●rb ∝ FITC (1: 750, 500 & 2k): Note that the original plan called for 1: 750, 1500 (= 1.5k) & 2k, so only 750 and 2k dilutions mounted. Both worked/looked great. However, at 1:2k there appeared to be some fibers better labeled than others. Did not make comparisons needed to tell whether this was due to concentration, or fiber size (or similar). To make best determination, further comparison of 1:750 & 2k groups needed, as well as comparison of ∝ FITC with SAvidin-labeled NT. However, the 1º has been in solution at 4ºC since July 2010, so I took my best guess and aliquoted the 1º in order to store it at -70ºC.  Aliquots contain 15 µl, which makes 15 ml at 1:1k, or 30 ml at 1:2k 

TRIS-Buffered Glycerol, pH 7.2

Recipe: Tris-Buffered Glycerol (TBG), 5% n-Propyl Gallate (nPG), pH 7.2

To make 10 ml TBG 7.2:
● Add 0.5 g nPG to 1 ml 2M Tris, pH 7.2*
● Add nPG/Tris to 9 ml glycerol.
● Mix well, aliquot and store at -20º C

For 50 ml TBG 7.2:

● Add 2.5 g nPG to 5 ml 2M Tris, pH 7.2*

● Add nPG/Tris to 45 ml glycerol.

● Mix well, aliquot and store at -20º C

Note:

1. 2M Tris = 1.6 g Tris HCl + 0.134 Tris Base in DI (blue-cap or better). Note that a calomel electrode is needed to accurately measure Tris pH
2. nPG goes readily into solution at about 60ºC. Use a water bath heated to 65 - 70ºC to allow time to vortex nPG solution and add to glycerol without precipitating out of solution (and have glycerol measured and ready...)
3. 500 µl aliquots are enough to coverslip 6 to 8 slides depending on coverslip size, and yield at best 20 aliquots from 10 ml. Scintillation vials and 50 ml centrifuge tubes work well for making larger volumes
4. nPG is an antifade agent that slows bleaching of cyanine and alexa dyes, but can be omitted if desired.
5. Recipe was adapted from Confocal Microscopy for Biologists (Hibbs, A.R., 2004; Springer).

Results & Rationale
Originally tested 8/16/2012 as a means of limiting the excitability of Fluorogold (FG) by wavelengths longer than UV, or at least < 488 nm to keep it out of the green (and red channels). 

Two TBG mounting media at pH 7.2 and 7.6 were compared to the normal bicarbonate (KHCO3)-buffered glycerol (BBG, pH 8.6), and tested on FG-injected as well as green- or red-labeled peptide IHC.

Both TBG media both reduce the number of FG-labeled neurons visible in the green and red channels compared to BBG without attenuating the FG signal or interfering with normal peptide IHC. However, TBG, 7.2 was clearly more effective, eliminating all neuronal labeling in both channels. At the injection site, FG labeling was clearly visible in the green and red channels when mounted w/either BBG or TBG 7.6, but faintly visible in the green channel only w/TBG 7.2. 

New PBS-PFA Recipe

The PBS-PFA tested on 9/4/2012 seemed to work fine, but for the future we are changing to a more common variant of Sorensen's phosphate buffer, based on recipes from  UC Berkley Biological Imaging Resources and Wikipedia—Phosphate Buffered Saline, and adapted for use with anhydrous Na2HPO4 (Berkley used 7●H2O, and Wikipedia 2●H2O)

PBS, pH 7.4: 

For 1 liter of 20X Stock use:

KCl                                         4 g

NaCl                                   160 g

Na2HPO4 (anhydrous)          22.9  g

(Sodium Phosphate Dibasic)

KH2PO4                                  4 g

(Potassium Phosphate Monobasic)

For 1 liter of PBS-Working, add 50 ml 20x stock to 950 ml DI

4% PFA in PBS, pH 7.4:

• For 1 liter of 4% Paraformaldehyde (PFA) in PBS, add 40 g PFA per liter of working PBS 
• For 4 liters of 4% PFA in PBS, add 200 ml 20x PBS Stock to 3800 ml DI.
• Heat to 60 - 70º C (Do Not Exceed 70ºC) and add 160 g PFA. Add NaOH (0.5 g/l) if needed to get PFA in solution. 
• Filter the PFA, with coffee filter, when cool enough to touch
• Move filtered PFA to 4º C overnight, or use salted-ice bath if perfusing the same day 
• Measure pH of cold PFA (4 - 8º C) and adjust pH to 7.4 with a few drops of HCl (12 N) or NaOH (1 to 5 N) if needed. 
• Prepare Sucrose Postfix (10 to 20% Sucrose in cold, pH'd 4% PFA). 
• Postfix for one animal = 11.5 g sucrose in 75 ml PFA (= 15%). For 3 animals = 35 g sucrose in 225 ml PFA; and postfix for 4 animals = 46 g sucrose in 300 ml PFA.

Perfusion Protocol:

Perfuse animals with normal saline (9g NaCl/liter) for about 5 min at a pump setting of 1.1 (= 40 ml/min) and then switch to PFA. When rigor sets in (or about a minute, whichever comes first), reduce pump speed to 0.8 (= 25 ml/min) for 20 min. Total volume of PFA perfused should be just over 500 ml. Remove the brain and place in cold sucrose postfix for 18 to 24 hr.

Protocol- Immuno Log Entry

Date                Antibody Used (dilution; tissue)

• Transfer tissue to 24 well nets
• Rinse out cryo w/ 50mMTBS7.2 X # rinses, time of each rinse
• LTBS= ?mL 50mMTBS7.2 + ?µl 25%T + ?mL NDS
• 1˚-
• 1 aliquot= ?µl 
• 1:?k= take ?µl 
• add ?ml LTBS= ?ml 1:?k
• use ?mL for 1:?k
• 1:?k= take ?mL 1:?k
• add ?mL LTBS= ?ml 1:?k
• use ?mL for 1:?k
• 1:?k= take ?mL 1:?k
• add ?mL LTBS= ?mL 1:?k
• Pre-incubate/Preabsorb for ? min
• Transfer to 1˚ -> 4˚ @ time

Date

• Rinse out 1˚ @ time
• total time in 1˚
• 50mMTBS7.2 X # rinses, time of each rinse
• Sort sections for secondary reaction
• LTBS= ?mL 50mMTBS7.2 + ?µl 25%T + ?mL NDS
• 2˚-
• name the secondary being used at what concentration
• calculate the dilution of the secondary
• Pre-incubate/preabsorb for ? min
• Transfer to 2˚ for ? time
• Rinse out 2˚ at @ time
• total time in 2˚
• 50mMTBS X # rinses, time of each rinse
• 3˚-
• name the fluor label being used at what concentration
• calculate the dilution of the label in 50mMTBS7.2
• Transfer to 3˚ at ? time
• Rinse out 3˚ at ? time
• total time in 3˚
• 50mMTBS X # rinses, time of each rinse
• Transfer sections to 6-well trays @ time to await mounting

Protocol- Anti-FITC Immuno Template

Schedule for FITC Immuno Round Completion:

Single labeling:
Friday- Sections into Primary (5x5min rinses, 30min pre-incubation/pre-absorption)

Monday- FITC(DK)anti(RB/MS) 1:500 in LTBS (5x5min rinses, 30min pre-incubation/pre-absorbtion,  
             2-3hrs in 2˚
             Alexa488(RB)anti(FITC) 1:500-2k? in TBS ~36hrs

Wednesday- Cy2(MS)anti(FITC) (1:1-2k) in TBS ~2-3hrs

Double labeling: 
Friday- Sections into Primary (5x5min rinses, 30min pre-incubation/pre-absorption)

Monday- Biot(DK)anti(RB) 1:500 in LTBS (5x5min rinses, 30min pre-incubation/pre-absorbtion, 2hr)
            FITC(DK)anti(MS) 1:500 in LTBS (5x5min rinses, 30min pre-incubation/pre-absorbtion, 2hr)
            Cy2(MS)anti(FITC) 1:500-2k in TBS ~36hrs

Wednesday- Cy3SAv 1:500 in TBS ~2hrs
                 

OR

Friday- Sections into Primary (5x5min rinses, 30min pre-incubation/pre-absorption)

Monday- Biot(DK)anti(MS) 1:500 in LTBS (5x5min rinses, 30min pre-incubation/pre-absorbtion, 2hr)                   
               FITC(DK)anti(RB) 1:500 in LTBS (5x5min rinses, 30min pre-incubation/pre-absorbtion, 2hr)
               Alexa 488(RB)anti(FITC) 1:500 in TBS ~36hr

Wednesday- Cy3SAv 1:500 ~2hrs
               (5x5min rinses)
                 

Recipe- Perfusate

Tested on 9/4/2012 (pH when 4˚ ~7.5)

To make 1 Liter PBS:

1Liter Blue Cap DI Water
NaCl 6.8g
Na2HPO4 1.5g
NaH2PO4 0.43g

add 4% Paraformaldehyde (40g/L)