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Immunohistochemistry (IHC) / Staining for acidic pH in formalin fixed tissue
« Last post by JMeehan on August 03, 2017, 11:57:14 AM »
Does anyone know of a way to stain for acidic pH areas in sections cut from tissue that has already been formalin-fixed and embedded in paraffin? I know there are methods to assess pH in tissue that has not yet been formalin-fixed, but I am not aware of any that can be used to give an idea of the pH in different areas after the tissue has been fixed. Any help would be greatly appreciated. Thanks, James.
In the past I have used neutral buffered formalin to fix mouse mammary glands prior to IHC and/or IF. Many examples in the literature also specify NBF. However, our lab routinely uses 4% paraformaldehyde for other organs. Does anyone know why I should choose one over the other? Is there something particular about mouse mammary glands that favours NBF?
Neuroscience / Quanitification of IHC images
« Last post by GAnthoni on May 17, 2017, 03:34:21 PM »
Hey team, hoping for some help here.
The images look great, but I'm running into a roadblock for quantification of neurites in hippocampus, any suggestions?
Details: I have several coronal rat brain slices (50um thick) which have three abs on them (primaries added as a cocktail, anti- TH, p-75, and ChAT) HRP- conjugated secondaries added individually and washed (anti- mouse, goat, and rabbit) each with an amplification step (TSA --> FITC, TRITC, and Coumarin) [these are super old animals]. I used a Nikon A1 scanning confocal to get ~.8um sections (from 11-20ish sections each) at 20x. In order to make sure results are quantifyable, the exact same microscope settings are used to capture each image stack.
Ive used ImageJ to split color and then manually adjust brightness and contrast of each stack such that adjusted levels match one another for each color. (this has been optimized to a "one size fits most" level). This is the step I believe may be causing problems, but let me continue.
This is followed by subtracting the background with a standardized "rolling ball" algorithm. Finally, the image is made binary using the auto-threshold function “Moments” (Tsai’s method attempts to preserve the moments of the original image in the threshold result; Tsai, 1985). From here the ROI boundary is thrown on and "analyze particles" is used to quantify objects by %area.
The problem is that when I use these "universal" brightness/contrast levels across all stacks of the same color (in the same region of the hippocampus) I get one result that looks good, but seems to hide a substantial number of neurites that may be important. However when I try to include the multitude of "missing signal" by creating images based on "relative levels" (in which each stack gets a custom brightness/contrast modification), the results are strikingly different. While I trust the universal levels to be comparable in regards to absolute levels of antigen present (necessary for results) I feel like there is a large amount of information that is missing (number of antigen-positive axons). I have come to the general conclusion that the %area obtained from the "universal" or "absolute" levels is a reflection of just that, the absolute levels of antigen being detected (of course this doesn't account for linear ranges above the detection threshold...) and that by modulating the brightness/contrast individually for each stack I am better able to detect number of antigen-positive structures.
Am I right to think this? Is there a better way to go about accurately assessing actual levels of these antigens? (we'll be doing Westerns too, but the cytoarchitectural information is critical to understanding potential mechanisms of action).

General Discussion / <share>Principle and Protocol of Co-Immunoprecipitation
« Last post by MCaroline on April 27, 2017, 02:03:49 AM »
Co-Immunoprecipitation (Co-IP) was developed from the immunoprecipitation technique with which Co-IP shares the fundamental principle of the specific antigen-antibody reaction. Immunoprecipitation(IP) technique is used for isolate individual protein. Using an antibody that is specific for a particular protein, the target protein could be isolated out from a crude lysate of a plant or animal tissue or other biological regent.

Immunoprecipitation of intact protein complexes is known as co-IP, which could pull the entire protein complex out of solution and thereby identify unknown members of the complex. Co-IP is a powerful technique that is used regularly by molecular biologists to analyze protein–protein interactions.

Prepare lysate form cells or tissue samples which express the target protein is the first step of the co-IP. In order to preserve the intact of protein-protein interactions, the lysis buffers should use non-ionic detergents (e.g., NP-40, Triton X-100). Then the target proteins are captured by specific antibodies from total lysate. The resultant immunocomplexes (composed of antibody, protein of interest (antigen), and antigen-associated proteins) can be precipitated using a resin (e.g., agarose, sepharose, or magnetic beads) that is conjugated with IgG-binding Protein A/G. The third step is washes. Irrelevant, non-binding proteins, antigens and any proteins that are bound are eluted by series of washes. Then, the bound proteins which eluted are analyzed by SDS-PAGE/immunoblotting and/or mass spectrometry.

As mentioned above, proper experimental conditions must be determined for each protein-protein interaction. Selection of an optimal lysis buffer and immunoprecipitation antibody are the two most important aspects for the success of a co-IP experiment. To overcome these problem, the protein of interest is often fused with an epitope tag (e.g., flag, myc, HA, his, V5), or a fluorescence protein (e.g., GFP, DsRed) at the terminus of the protein, and ectopically expressed in cells. Antibodies for these “tags” that are compatible with IP/co-IP have been well developed and are commercially available from multiple manufacturers.


Transfection of plasmids expressing proteins of interest. Follow the transfection protocol.
48h after tansfection, harvest the cells using a cell scraper and pellet the cell by centrifugation at 1000g×6min at 4℃.
Resuspend the cells tenderly by pre-cold PBS. Centrifugation at 1000g×6min at 4℃ and discard the supernatant.
Repeat Step 3 for three times.
Resuspend the cells tenderly by pre-cold lysis buffer (add protease and phosphatase inhibitor prior to use).
Incubate the samples on ice for 30 min. Convert the tube up and down during this period.
Centrifuge at 15,000 g×10 min at 4℃ and transfer the supernatant to a new 1.5 ml tube.
Measure protein concentration.
Using a tip to transfer the IgG-crosslinked resins to a EP tube containing 1ml PBS.
Pellet the resin by centrifugation at 6000 g×30 s, and aspirate the supernatant.
Repeat step 10 for three times.
Resuspend the resin in pre-cold lysis buffer.
Transfer 750μg of total protein to a new tube and adjust the volume to 200μl with pre-cold lysis buffer.
Add 50μl of the prepared resin.
Incubate the tubes on a tube rotator at a slow speed for 1 h at 4 ℃.
Centrifugation at 6000 g for 30 s at 4 ℃ and transfer 200 μl of the supernatant to a new tube. Save the pelleted resins to use as negative controls.
Dilute the antibody to proper concentration with lysis buffer.
Add diluted antibody to lysate prepared in step 16 and incubate the tube on a rotation 1h (or overnight) at 4 ℃.
Prepare Protein G-immobilized resin as instruction of step 9-12. (Protein G is often considered a more universal IgG binding protein than is Protein A, but different species, and subtypes of species, do vary in their binding to these proteins.)
Add 50 μl of the prepared resin to each reaction tube in step 18 and incubate on a rotator for 1h at 4 ℃.
Centrifugation at 6000g×30 s at 4 ℃. Transfer 100 μl of supernatant to a new tube and aspirate the remaining supernatant.
Resuspend the resin with 500 μl pre-cold lysis buffer, centrifugation at 6000g×30 s at 4 ℃, and and aspirate the remaining supernatant.
Repeat step 22 for three times.
Resuspend the resin-bound immune complexes in 20 μl of 2 Laemmli buffer, boil for 5 min, and analyze by SDS-PAGE/immunoblot analysis.

Hello everyone,

I am a radiation oncologist from Turkey and I am studying neurogenesis on hippocampus of rats. Currently my research partner in histology and I are having difficulties with staining sample tissues using CD68 (Santa Cruz), doublecortin (Santa Cruz) and Ki67 (Abcam ab16667) markers  :(

So far we have taken the steps below but unfortunately that didn't yield selective staining. Could you spot anything we possibly may have done wrong?

We also tried skipping step #8. The antibody was diluted 1/100 and we waited overnight at +4°C. Most parts of the tissue were stained but there was no specific staining.

To see if the antibody is working, positive control stain was made in the intestinal sample. The antibody was diluted 1/100 and stood overnight at +4°C. It stained.

After sacrification, we performed intracardiac perfusion fixation, and the tissues were embedded in paraffin within 2 weeks. We have not encountered any problems with cutting tissues with the microtome.

1. The brains of rats were embedded in paraffin after perfusion fixation procedure
2. Sections with 4μ thickness were taken
3. Deparaphinization for 1 night at incubator (at 60ᵒC)
4. Xylene for 30 minutes
5. Dehydration in graded series of alcohols (100%, 90%, 75%, dH2O)
6. Antigen retrieval for 30 min at 37ᵒC trypsin or 0.01M citrate buffer 3x1min, 3x5min (all possibilities have been tried)
7. PBS for 3x5min
8. 3% H2O2 in Methanol x5 min, 1% H2O2 in Methanol x15 min, 3% H2O2 in PBS x5 min (all possibilities have been tried)
9. Blocking solution x 5 min
10. Stained with: DCX (sc390645) diluted 1/100 and 1/50 with PBS, Ki67 (ab16667) diluted 1/100 and 1/50 with PBS, CD68 (sc7084) diluted 1/100 and 1/50 with PBS, 2 hours at room temperature, 1 hour at 37ᵒC or overnight at +4ᵒC (all possibilities have been tried)
11. PBS for 3x5 min
12. Seconder antibody for 10 min
13. PBS for 3x5 min
14.Enzyme-labeled avidin-biotin complex for 10 min
15. PBS for 3x5 min
16. DAB x 1 min, 2 min
17. Washed with dH2O
18. Hematoxylin for 1 min or no hematoxylin staining (all possibilities have been tried)
19. dH2O, 80%,90%, 100% alcohols and xylene
20. Closed with Entellan

We would be grateful if someone can point out where we possibly made a mistake? Thank you in advance.
Immunohistochemistry (IHC) / IHC and IF combination
« Last post by SRobert on March 01, 2017, 10:52:32 AM »
Does anyone know of a protocol for staining the same (paraffin-embedded) slide for one antigen for IHC (Vectastain ABC method) and another antigen with IF? I've been told it is possible but cannot find a protocol.  Thanks for your help!
Biochemistry / Teduglutide
« Last post by SBella on February 28, 2017, 02:00:05 AM »
Teduglutide differs from natural GLP-2 by a single amino acid: an alanine is replaced with a glycine. This blocks breaking down of the molecule by dipeptidyl peptidase and increases its half-life from seven minutes (GLP-2) to about two hours, while retaining its biological actions. These include maintenance of the intestinal mucosa, increasing intestinal blood flow, reducing gastrointestinal motility and secretion of gastric acid.
Biochemistry / Antide
« Last post by SBella on February 28, 2017, 01:57:45 AM »
Antide acetate (Ac-AA10-NH2) is an LHRH antagonist and represses LH and FSH release from the pituitary gland. It shows a high antiovulatory activity and releases negligible histamine.
Immunofluorescence (IF) / Re: Staining on only the periphery of tissue
« Last post by QChong on February 13, 2017, 05:06:52 PM »
someone suggest me to cover the antibody drop with a parafilm to avoid edge effect. may be you can try. good luck.
Immunofluorescence (IF) / unmask by proteinaseK
« Last post by QChong on February 13, 2017, 04:56:41 PM »
liver staining by CD31- rat anti mouse antibody(Abcam Catalog Number: ab28364), only the edge and around of the vessels of the liver can be stained clearly(see the attachment).
the liver to be fixed are no more than 3mm, fixed by zink fixation buffer for 72h,
proteinaseK is 20ug/ml, 30min@RT, followed by Permeabilization. 
Could you please help me to find how to stain the tissue evenly? thanks.
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