This is book S, which begins on Thursday, December 16th, 1965, and runs through about April of 1965. This is the beginning of quite a large number of experiments which were done that involved the thymus and cultured cell primarily driven by these ideas on the antibody virus for what it was worth. It begins by some experiments with Alice Claflin trying to find good ways of locating lymph nodes and also looking [00:01:00] what tissues take up antigen. So four animals were injected in different ways, IV or intraperitoneally or into the footpad. And then their tissues were taken overnight on Saturday, December 18th, where my writing is there alongside Alice’s, showing the order of blackness of tissues after overnight fix, where the score is evidently number 10 is meant to be high. Because starting with four pluses it was crossed out and 10 is put in. And the liver and spleen in animal 47 for example which had received [00:02:00] 0.1 ml of india ink intravenously is very heavy as is the spleen and so on.
Continuing this type of experiment. With also some ideas that involved treatment with cortisone there on January 3rd and so on for several days thereafter. All in Alice’s writing. None of it in mine. Continuing in this book then with Alice’s. With a [00:03:00] gel that was run on Thursday, January 6th, which Alice ran backwards apparently. So these are all essentially Alice’s experiments going on for several pages until my next entry is on Thursday, January 13th looking at animals some of which had already died in various groups.[00:04:00] These experiments are of various forms. The spleen was plated and the thymus was plated into various tissue culture things which are no longer clear to me or written very clearly in the book. Continuing in this general manner for [00:05:00] many more pages with a gel of haptoglobin on page 34 but no real documentation of what it was about. This book so far is almost entirely Alice’s writing. The next note of mine [00:06:00] is on February 9th, 1966, page 41. Just a comment on the mean and no difference from the usual control, etc.
So seems to have been a period when I was not doing any direct experiment. It could well have been a period when I was teaching. That is not apparent from the notebook. Page 45 indicates something about what’s going on because a TVB gel looks with my notation on it, gamma globulin [00:07:00] fractions for something. Smithies and Bence-Jones purified samples for Edmundson. Allen Edmundson. Bence-Jones from Allen Edmundson’s Bence-Jones protein.
Was paying attention as judged by the plot on page 46 with my notes on it of [00:08:00] antibody titers presumably. So the next really solid page from me is on page 53, February 25th, Friday. Recovery experiment. Late phases of the response. Aim to check for possible information transfer experiment. So it is this antibody virus business which is still going on in my head. Aim to check if animal two can supply effector cells for animal one damaged by lethal X-rays. [00:09:00] So the idea was to X-ray the animals and prevent them from dividing, which I remember quite distinctly doing.
And this is the plan on page 53. So a test of X-ray sensitivity of 60-hour plaques to complement S46 and Alice labeling test with antigen 1 is on page 55 with a scheme there of what is going to be done. X-ray [00:10:00] 1,000 rads 40 minutes maximum 240 kilovolts 15 milliamps. But bacterial contamination of the plates so the experiment had to be repeated. Here it is again on Tuesday, March 15th. Again bacterial contamination query of the plates. I think I remember that now. It was that we had dried — we had prepared the clostridium and if I remember correctly the bacteria were [00:11:00] really rather prevalent in the lab, bacterial spores, and gave us a lot of trouble. So here is Thursday, March 3rd. Alice has the titer book AE123. X-ray spleen. Normal spleen, so on, so forth.
And here on March 9th, Wednesday, specific thymus type. The general hypothesis that some cells have recognition sites in surface should be testable. Take unrelated antigens A and B, etc., etc. [00:12:00] Ideas and some comments on other items. The availability from Harold Deutsch of [Nevin?] multiple myeloma conjugates for gamma globulin, etc.
Conjugating [Nevin?] multiple myeloma protein with fluorescein isothiocyanate. The experiments with the thymus were made difficult obviously on page 67. Rough tests with thymocyte show too much general fluorescence. [00:13:00] These are plates of red cells looking for plaques. Back under control on Thursday, March 10th. [00:14:00] And here’s mention of [Nancy?] again on page 71, March 11th, [Nancy?] read Friday afternoon, and I read on Saturday morning.
Trying to find out if there were antibody-producing cells in the thymus. It looks as if that’s what was going on. High [00:15:00] voltage electrophoresis test of [insertion?] of the samples Thursday, March 18th. [Poor?] result. [They’re just?] terrible. Normal [cook?] looks quite promising. Rather pretty set of gels the following page. On [Dick Kondi?] hemocyanins. Probably done for something to check. Monday, March 21st, local X-ray protection. Chloral hydrate used to sedate the animal and then [00:16:00] normal respiration, etc., gasping respiration on one animal. Chloral hydrate test again. And this is a rather interesting set of diagrams because I very well remember the result of this. This is page 87. Probably March 21st, Monday. The chloral hydrate and then [00:17:00] X-ray to protect different parts of the animal. So I made a lead block which I could put over the thymus area or lead over the pelvis and holes in the reciprocal place. And which this one is I’m not sure at this point. But I do remember the result, which is that when I irradiated the thymus I didn’t see any change in the thymus but when I irradiated the rest of the animal the thymus was very badly damaged, which I now know means that there was a cortisol lysis induced by the whole body irradiation. I might have made [00:18:00] quite a useful contribution to this effect if I’d understood that experiment. But it goes on on page 93 where in the interim there are some photographs of what a plaque looks like with a comment that with whole blood it’s difficult to find possible plaques. And there’s an image there. But with washed cells it’s possible to find a good plaque. Judging from later ones I know it was (inaudible) so the limited X-ray experiment talked about on page 93.[00:19:00] And 5 of the 10 animals which were unprotected were dead by Monday morning although all were alive Friday afternoon. No deaths at all in the six animals where the thymus and the [broom?] were protected. So here are the charts of what happened. And plating presumably thymus. Presumably spleen. Images of the plaques that were obtained. Not very convincing but [00:20:00] they are plaques.
Some tests of altering the medium on March 28th and continuing with magnesium test on the 29th. DEAE-dextran made up without any written rationale. Still thinking about local X-rays and made up five shields. Here is the one with the hole that covers a large part of the body with a hole over the thymus. [00:21:00] And five rather bigger small pieces for local irradiation and protection of the thymus. So here was the result, which was the surprising result on the left, page 104, that with — I had thymus only or the rest of the body. And the doses of irradiation. And the experiment was to determine whether the thymus decreases in weight more extensively when it is irradiated or when the rest of the animal is irradiated. And the [00:22:00] results are very surprising because the animals with less rest of the body protected had better thymuses I’m pretty sure than the ones where the thymus was protected. Although it doesn’t say that specifically. Except it does say these results are very surprising. Must repeat the experiment with better still shielding. [Triangles?] the other way up and locate the thymus by drawing on the surface, etc. Was quite puzzled by this opposite effect from what I expected.[00:23:00] None of this was ever published. A note by presumably Alice on Wednesday, April 6th that both of the remaining rest of the body animals are dead. Means irradiation of the rest of the body. Although they had been alive earlier. But those in which the thymus was irradiated were OK. [00:24:00] Some transferrins as a relief. [Jeannette Sams’s?] transferrins. Gel on Friday, April 8th. Check of the shielding in the X-rays were tried with a dosimeter design. Results showed much backscatter. So redesign the shielding. [00:25:00] So some images of what the thymus looked like on pages 118 and 119. Changes in the cellularity. By thymus only. That’s protection. Thymus only has a large amount of tissue loss of thymus cells. But if the rest of the body was irradiated — was protected — there was no change in cellularity in the thymus. [00:26:00] Or at least not (inaudible) general and progressive but slower decrease in cellularity. Possibly [some active false ILF?] whereas on the thymus only protected very rapid and clear decrease in cellularity.
More images on the following pages, 124 and 125. No clear-cut changes in germinal center in the spleen with thymus only being X-rayed. A comment, use the lymph node for future work. Marked atrophy of the lymphoid elements when [00:27:00] the rest of the body was irradiated again.
Staining difficulties as always for me because of the problems of the color vision. Need to have more orange G and eosin Y staining and less toluidine blue. Try using only orange G and decreasing the amount of toluidine blue. And messing around with stains. Still a problem in my current work.[00:28:00] Some rather obscure comment on page 130 [LPB537?] cDNA expression in hepatoma. Martius yellow. I remember Martius yellow. Talked about on page 133. [00:29:00] I like the stain. Yeah. And with a comment, however, that best yet is PA, picric acid, 10 minutes and toluidine blue for 20 seconds. And differentiate. The usual business of histology trying to find the right way to stain things to see what you want to see. Thursday, April 14th, this is the best, toluidine blue for 20 seconds and then MYA, Martius yellow acid, generally good. But still too green is the comment. More staining tests on page 137. And preparation of dyes on page 139. I used to have a tremendous collection of dyes. [00:30:00] How to make Martius yellow solution. Whether wetting agents would help. And then back to splitting solution on Sunday. Two samples of [Jeannette’s?] transferrins. [00:31:00] Also on the following page the same sort of thing. Tuesday, April 26th, page 151. Stain tidy up. The result on the left-hand side describing the results with the Martius yellow stain and toluidine blue with a big ellipse excellent, like staining. And that ends this book, page 152.