Bugen Hu        August 7, 2009

 

Legend for the lung images by using my unpublished protocols of Y FISH and immunofluorescent staining contained in the CD

 

All the images are coming from the original images when I examined the slides under fluorescent microscope, I did not do any “art work or edit” on any of such images.

 

Amplification: 40X

 

1. Wild type male mouse lung paraffin section labeled with Y FISH, anti-SPC (in-house guinea pig anti-spc serum from Jeff’s lab), and anti-CD45/F4/80. Signals labeled as the following: Rhodamine for Y (intense pink dots in the DAPI counter stained blue nucleus), Alexafluor 488 for SPC (green in cytoplasmic part), Texas-Red for CD45 and F4/80 (orange red on cell membrane). If the authors could really get experimental protocols of Y FISH and immunofluorescent staining (anti-SPC, and anti-CD45) work, this image should be positive control image for the FASEB paper Fig 2A. This image can show the people, including the peer-reviewers, and readers how well the experimental methods (protocols) are, and what should be the “positive markers” for each labeling (Y/SPC/CD45), and these “positive markers” right locations on the slide and in the cell. The most important information of this image should show is that after all labeling is finished (Y FISH, and immunofluorescent staining), as a whole picture what really looks like (Y signal: what percentage of cells is labeled as Y positive, SPC signal: in the physiological locations of lung type II cells (on the alveoli) how well the type II cells are labeled as SPC positive, CD45 signal: whether or not membrane of leukocyte can be labeled as CD45 positive). In this image: a. The whole structure of alveoli is clearly shown up at normal exposure time due to autofluorescence of the tissue itself, b. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative (detection sensitivity of Y FISH protocol, but this image is still extremely good image in terms of Y FISH and SPC staining detection sensitivity if you have doubt about it, you can do literature search on internet to see if there is any image of lung paraffin section labeled with Y FISH and immunofluorescent staining in any published papers is better than this image). c. At right physiological locations of type II cells there are a lot of SPC and Y doubt positive cells (definitely certain percentage of type II cells must be stained as SPC negative because I don’t think that my protocols can reach 100% detection sensitivity for anti-SPC immunofluorescent staining). d. There is no cell being SPC and CD45 double positive (in this field there is no obvious CD45 positive cell).
2. spcko male mouse lung paraffin section labeled with Y FISH, anti-spc, anti-CD45, and anti-F4/80. Compared with the image of wt male control, overall spcko has much stronger autofluorescence (the whole alveoli structure is clearly shown up as very strong green autofluorescence even exposure conditions I used is the same as I used for wt male control section). For Formalin or PFA fixed tissues, lung is one of the tissues with the strongest autofluorescence, another one is GI tissue), and the alveoli have more “fibers” than the alveoli of wt male mouse. In this image: a. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative. b. No single cell is labeled as SPC positive, no single cell even really looks like the SPC positive cell in the wt male control image (even there are some cells with green in their cytosplasmic part “look-like positive”, but this so-called green signal is the same as that of alveoli “fibers” but different from the real SPC positive staining in the image of wt male slide, it is very easy to tell difference between signals of SPC specific staining and green autofluorescence when compared with the real positive cells in the wt male control). c. In this field there is obvious CD45 positive cell. If the authors’ protocols for Y FISH and anti-SPC really work, at least double-labeling image should be presented as the image of spcko male control Fig 2B for the paper FASEB 2007. Only using above mentioned wt male and spcko male multiple labeled sections can really function as controls to show: how well Y FISH protocol works in both wt male and spcko male tissues (whether or not Y can achieve similar detection sensitivity in both tissues), and clear-cut difference between wt male and spcko male sections in terms of SPC staining, and physiological distribution of SPC positive labeled type II cells in the lung.
3. Paraffin section from the lung of spcko male recipient of wt female bone marrow transplantation (model mouse ID: spc-14) labeled with Y FISH, anti-SPC, anti-CD45, and anti-F4/80. This image shows chimera characteristics of such model. In this image: a. Whole structure of alveoli is clearly shown up (as green autofluorescence of thick “fibers”). b. Most nuclei of CD45 negative cells are labeled as Y positive, but there is still a small percentage of such cells being labeled as Y negative. c. There are several obvious CD45 positive and Y negative cells (leukocytes derived from donor bone marrow cells, that is, so-called “engraftment”). d. There is no single cell being SPC positive, in terms of SPC specific staining the situation is the same as I mentioned for the image of spcko male control mouse. If the authors actually did such model, and their published protocols of Y FISH and immunofluorescent staining really work, by using such model to identify and prove that in vivo donor bone marrow cell can derive into type II cell by the mechanism of fusion with recipient cell, this is the image the authors should present as the Fig 2C in the paper FASEB 2007 (at least to show people that the image is really coming from lung of such model, their image Fig 2C logically can come from any mouse lung, and so-called “SPC positive” could be anything including autofluorescence of cytoplasmic part). This is the image (spc-14) containing the “two look-like positive cells with double positive” Dr. Diane Krause really wanted and put huge pressure on me trying to force me change my own judgment call on the two cells from negative to positive cells (Y and spc double positive). When I put the images of wt male and spc-14 side by side, and asked her: “Even when you compare image of the two cells with image of positive control (wt male), the color and staining pattern is same or not?” She said: “not same, but stem cell derived type II cell is not as same as real type II cell.”  And I repeated my principle by saying: “As PI you can make your own judgment call on my slides any way you want, and it is perfectly fine with me, but I only hold responsibility for my own judgment call.” Then she forced me to trust and accept “the results” form the paper (FASEB 2007), after I firmly refused she became so emotional and angry with me. Finally she sent an email to Dr. Erica Herzog to ask her as blinder to examine my slides under microscope and ask Dr. Erica Herzog to show us electronic images of anti-spB the authors used for the paper FASEB 2007 (the facts are: a. Erica never showed us any images of anti-spB she only said that she needs to find such images of anti-spB from the computer., but she never showed us any anti-spB image (I don’t think that she has such anti-spB images). b. No single image of anti-spB was presented in the paper. c. In the inventory boxes for immunofluorescent reagents in Dr. Krause’s lab I had never seen any vials of anti-spB antibody before February, 2008. I ordered my anti-spB from Chemicon in February 2008. I have the email to prove). On March 6, 2008 after looking at my slides under fluorescent microscope, Dr. Erica Herzog sent me that “famous” email.
4. Normal wt male mouse lung cytospins slide labeled with Y FISH, anti-SPC, and anti-CD45. In this image: a. Most of nuclei of cells are labeled as Y positive, but a few cells being Y negative, for example, one cell at the bottom showing most part of its nucleus. b. There are three cells being labeled as Y and SPC double positive (one is located at left of the image, two are located in the middle). c. At the left of the image adjacent to the Y SPC double positive cell there are several cells being labeled as Y positive and with yellow-green autofluorescence in their cytosplamic parts (such Y positive cells with yellow-green autofluorescence in cytoplasmic parts are common on the male mouse lung cytospins slides labeled with just Y FISH without any other immunofluorescent staining, such cells with yellow-green autofluorescence in their cytoplasmic parts are common on the mouse lung cytospins slides without any labeling, that is, blank control for immunofluorescent staining if the samples are fixed with Formalin or PFA). d. There is a cell labeled as Y positive and look-like CD45 positive located in up-right area of the image. e. There is no single cell being labeled as SPC and CD45 double positive. This image can show real clear-cut difference between signal of real cytoplasmic marker (here is SPC) immunofluorescent staining and signal of cytoplasmic autofluorescence on the mouse lung cytospins. If the images of Fig 4A, 4B (paper FASEB 2007) are really coming from the lung of the model spcko female recipient of male marrow, and the authors’ published protocols of Y FISH and anti-CK staining really work, for Fig 4A, and 4B, the authors should present the images like this one showing Y and cytoplasmic marker positive cells plus information of signal of X FISH in nuclei, and cells adjacent to the “identified positive cells”.
5. Normal spcko male mouse lung cytospins slide labeled with Y FISH, anti-SPC, and anti-CD45. In this image: a. Most of nuclei of cells are labeled as Y positive, but a few nuclei are labeled as Y negative, located at the bottom of the image. b. No single cell is stained as SPC positive, but there are a lot of cells with strong green autofluorescence in their cytoplasmic parts. c. There are several cells containing multiple dots of Y signal in its nucleus. And scientific fact is that every cell on the slide can only contain one molecule of Y chromosomal DNA, so this image can clearly demonstrate this scientific fact that one molecule of Y DNA can be detected as Y negative, one dot of Y signal, or multiple dots of Y signal in the nucleus by interphase Y FISH.

 

Summary: These five images of mouse lung samples (paraffin sections and cytospins slides) labeled with Y FISH and immunofluorescent staining show:

 

1. For Formalin or PFA fixed mouse lung samples, cytoplasmic autofluorescence is common and strong on both paraffin section and cytospins slide, but there is clear-cut difference or manifestation between specific immunofluorescent staining signal of cytosplasmic marker and cytosplasmic autofluorescence. Without showing the real image of real positively labeled control, people not having direct experience in this technique can be easily fooled by cytoplasmic autofluorescence especially when the authors just present images with a single cell or a few cells without “reference system”.
2. For both paraffin section and cytospins slide, my unpublished Y FISH protocols can achieve much higher detection sensitivity. But I can say here that even using my own protocols I have never achieved 99% detection sensitivity for either paraffin or cytospins slide, even not close to 99% positive (my standard is whole slide, not just one chosen image). By using my protocols or protocols that can achieve similar detection sensitivity of Y, dishonest people can easily make fake image (containing 20, 30, or 50 cells) showing 100% cells being labeled as Y positive by just “cutting off” single cell or a few cells that are labeled as Y negative from the image. For example, by using my protocols of Y FISH and immunofluorescent staining (edited image), for male sample I can generate the double-labeled images (at 40x amplification, containing 20, 30, 50, or even 100 cells) showing “100% Y detection sensitivity”, and beautiful immunofluorescent staining with preserved tissue and cell morphology by just “cutting off” a cell or a few cells labeled as Y negative from the image. If I was dishonest, and I need such logic premise: 100% detection sensitivity of Y FISH, I submit such images as results and claim that my Y FISH protocol can reach 100% detection sensitivity, definitely based on manifestation of “the edited image” this statement is “true”, but scientifically this image does not truthfully reflect the real experimental phenomenon of the slide because the Y negative cell or cells in the real image or whole picture were intentionally “cutting off” from the image presented (edited image).
3. What the real images of mouse lung sample should look like after multiple labeling of Y FISH and immunofluorescent staining.
4. In the nucleus of one normal male cell, one molecule of Y chromosomal DNA can be detected as Y negative, one dot of Y signal, or multiple dots of Y signal.
5. Physiological distribution of type II cells labeled as Y and SPC double positive on alveoli of the normal male mouse lung paraffin section. For any people if their protocols of Y FISH and anti-SPC immunofluorescent staining really work, and the primary antibody of anti-SPC really works on Formalin or PFA fixed samples, they should be able to produce the similar images from the normal male mouse lung paraffin section as real wt positive control image if they want to use Y and anti-SPC dual markers to identify donor stem cell derived type II cell from the tissue of spc knockout sex-mismatched model.
6. How important it is to have both positive and negative control slides and “reference system” on the same slide when experimental methods of interphase Y FISH and immunofluorescent staining are used to identify donor stem cell derived tissue type cells in the recipient tissues, that is, without the controls or “reference system” to compare with, the so-called “identified positive cell” in the electronic image could be anything but real positive cell. Because in this situation two different kinds of experimental methods, in situ DNA hybridization and antigen- antibody interaction (immune staining) are used, and none of the methods can achieve 100% detection sensitivity and none of immunofluorescent staining can achieve 100% detection accuracy. The authors, Dr. Diane Krause, and Dr. Erica Herzog should know this principle pretty well because they have been using such methods to “identify” donor stem cell derived tissue type cells from the tissues of sex-mismatched models for years, and published multiple papers.

 

Feb. 4, 2009

 

Technical Information and Explanation of Erica Herzog’s Experimental Protocol for Y FISH on Bone Marrow Cytospins Published in the FASEB Paper (2007)

 

Here the most important step is missing: Denature the target DNA, i.e. Y chromosomal DNA (the size of this DNA is 97.5Mb, for C57 background mouse) to open the double stranded target DNA. The larger the size of the target DNA, the harsher condition is required to open the target DNA. To open the double stranded target DNA is precondition for any DNA hybridization experiment, otherwise the specific probe can not bind to the complement sequence on the target.

 

So-called Denature step in the authors’ protocol is 73C for 5 mins. Actually the major function of this step is not to denature the target DNA, but the probe, just like for normal DNA hybridization or classic FISH some people do denature the probe 1^st at 70-80C for about 10 mins before apply the probe to the target. Under this denature condition (73C, 5 mins) there is only certain percentage of cells their Y chromosomal DNA getting partial denature, i.e. double stranded DNA opened in some sections of the Y chromosomal DNA. That is, sacrifice the detection sensitivity for retaining the cells on the slide and cell morphology. Here the probe we used for Y FISH is a mixture of DNA, sizes from 100 to 500 nucleotides, its complement sequences on the target will cover the whole Y chromosomal DNA except for super high repeated sequences that will also appear in other chromosomes (the other name of Y FISH is whole Y chromosome painting).

 

When utilize Y FISH (identify the origin of donor) combined with immunofluorescent staining (identify the specific tissue marker) technique to identify the donor stem cell derived specific tissue cell in sex-mismatched models, the authors encounter technical dilemma: Y FISH requires very harsh condition which will destroy the tissue physiological structure and cells morphology including cytoplasmic and cell membrane component (like CD45) , while immunofluorescent staining is just antigen antibody interaction (here is protein and protein interaction) which requires mild condition. If just add these two different kinds of experimental methods together it will not work, regardless doing immunofluorescent staining 1^st or Y FISH 1^st because in order to identify donor stem cell (Y as marker of origin for donor or recipient) derived specific tissue type cell, after both experimental procedures the both signals need to be present on the same slide otherwise the mission is impossible. If the authors use the classic denature condition for Y FISH on tissue paraffin sections as they published, 1M sodium thiocyanate at 80C for 20 mins, then neutralized with 0.2N HCl at room temperature for 12 mins, after Y FISH for most cells only nuclei left. If the authors use such denature condition on cytospins, after Y FISH probably only a few nuclei left on the slide, or nothing left on the slide. And imagine even there is such biological event- bone marrow stem cell derives into specific tissue type cell (here is from BM to type II pneumocyte) happening in vivo, the frequency of such event is very low even based on the authors calculation (less than 1 per 1000). Since no immunofluorescent staining is 100% specific and accurate, the universal accepted standard to identify donor stem cell derived specific tissue cell is triple labeling: Y, tissue specific marker and CD45 in sex-mismatched models to exclude the leukocytes that can be present in any tissue.

 

How to solve this technical dilemma, it took me about 6 months to solve the technical dilemma above mentioned: interphase Y FISH combined with immunofluorescent staining while keeping the tissue and cell morphology normal. The strategy I used is to find the common window of experimental conditions that will fit for both Y FISH and immunofluorescent staining, then hard working of error and try. Even I say that I solved the technical dilemma, but for Y FISH my protocols cannot get 99% detection sensitivity on either paraffin section or cytospin slide. Definition of detection sensitivity needs to be defined by thousands of cells on the same slide, but not by a few cells of electronic image of very small field.

 

In order to test the detection sensitivity of Y FISH on bone marrow cytospins, and verify the authors experimental results generated by using such protocol, I just did Y FISH on bone marrow cytospins from the perfectly normal young wild type mouse by strictly following Dr Erica Herzog and Dr Diane Krause published protocol in their paper, FASEB vol.21 August 2007, p2592-2601. I will send the slide and a CD that contains the electronic images of such slides to the Dean’s Office, Yale School of Medicine.

 

Here are technical parameters:

Channel 1: DAPI to stain for DNA, blue color.

Channel 2: FITC, green color.

Channel 3: Rhodamine for Y signal, pink color (intense dot in nuclei).

Merged image: besides the Y signal, some yellow, green signals are coming from autofluorescence.

Amplification: 40x.

Rhodamine-conjugated anti-digoxigenin antibody (Roche), the same as Diane’s lab always used.

Digoxigenin labeled Y probe, the same as Diane’s lab always used.

IPLAB software is used to capture images.

 

After examine the whole slide under fluorescent microscope, my conclusion is: there is no way for the authors to obtain their experimental results as they claimed in their paper (FASEB 2007) because the Y FISH detection sensitivity is too low. So their results of Y FISH on bone marrow are just intentionally fabricated numbers, not scientific experimental results at all.

 

 

Bugen Hu

 

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